COMPOSITIONS CONTAINING A COMBINATION OF A CREATINE COMPOUND AND A SECOND AGENT

COMPOSITIONS CONTENANT DE LA CREATINE COMBINEE A UN SECOND AGENT

English Abstract

The present invention relates to the use of creatine compound and
neuroprotective combinations including creatine, creatine phosphate or analogs
of creatine, such as cyclocreatine, for treating diseases of the nervous
system. Creatine compounds in combination with neuroprotective agents can be
used as therapeutically effective compositions against a variety of diseases
of the nervous system such as diabetic and toxic neuropathies, peripheral
nervous system diseases, Alzheimer disease, Parkinson's disease, stroke,
Hungtington's disease, amyotropic lateral sclerosis, motor neuron disease,
traumatic nerve injury, multiple sclerosis, dysmyelination and demyelination
disorders, and mitochondrial diseases. The creatine compounds which can be
used in the present method include (1) creatine, creatine phosphate and
analogs of these compounds which can act as substrates or substrate analogs
for creatine kinase; (2) bisubstrate inhibitors of creatine kinase comprising
covalently linked structural analogs of adenosine triphosphate (ATP) and
creatine; (3) creatine analogs which can act as reversible or irreversible
inhibitors of creatine kinase; and (4) N-phosphorocreatine analogs bearing non-
transferable moieties which mimic the N-phosphoryl group.

French Abstract

L'invention concerne l'utilisation de composés de créatine combinés à des agents neuroprotecteurs, comprenant de la créatine, de la phosphocréatine ou les analogues de créatine tels que la cyclocréatine, pour le traitement des maladies du système nerveux. Les composés de créatine combinés à des agents neuroprotecteurs peuvent être utilisés comme compositions thérapeutiquement efficaces contre diverses maladies du système nerveux telles que les neuropathies diabétiques et toxiques, les maladies du système nerveux périphérique, la maladie d'Alzheimer, la maladie de Parkinson, les ictus, la chorée de Huntington, la sclérose latérale amyotrophique, les maladies des motoneurones, les lésions traumatiques des nerfs, la sclérose en plaque, la démyélinisation et les pathologies associées à cette dernière, ainsi que les maladies mitochondriales. Les composés de créatine qui conviennent comprennent (1) la créatine, la phosphocréatine et les analogues de ces composants pouvant agir comme substrats ou analogues de substrat pour la créatine kinase; (2) des inhibiteurs à deux substrats de créatine kinase comprenant des analogues structurels à liaison covalente d'adénosine triphosphate (ATP) et de créatine; (3) des analogues de créatine qui peuvent agir comme inhibiteurs réversibles ou irréversibles de la créatine-kinase; et (4) des analogues de N-phosphocréatine comprenant des fractions non transférables imitant le groupe N-phosphoryle.

Claims

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CLAIMS
What is claimed is:
1. A method for modulating a nervous system disease in a subject, comprising
administering to a subject a therapeutically effective amount of a
combination of creatine, a creatine phosphate or a creatine analog and a
neuroprotective
agent, such that a nervous system disease is modulated.
2. The method of claim 1, wherein said nervous system disease is modulated by
preventing the occurrence of the disease.
3. The method of claim 1, wherein said neuroprotective agent is a
mitochondria)
cofactor.
4. The method of claim 3, wherein said mitochondrial cofactor is 2,3
dimethoxy-5-methyl-6-decaprenyl benoquinone.
5. The method of claim 1, wherein said neuroprotective agent is an electron
transport chain regulator.
6. The method of claim 5, wherein said electron transport chain regulator is
nicotinamide.
7. The method of claim 1, wherein said neuroprotective agent is a spin trap.
8. The method of claim 7, wherein said spin trap is PBN.
9. The method of claim 1, wherein said neuroprotective agent is a cofactor for
normal cellular metabolism.
10. The method of claim 9, wherein said cofactor is carnitine.
11. The method of claim 1, wherein said neuroprotective agent is an
antioxidant.
12. The method of claim 11, wherein said antioxidant is vitamin E.
13. The method of claim 1, wherein said neuroprotective agent is a vitamin.

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14. The method of claim 13, wherein said vitamin is riboflavin.
15. The method of claim 1, further comprising administering at least one
additional
neuroprotective agent or creatine compound.
16. The method of claim 1, wherein said creatine compound is creatine.
17. The method of claim 1, wherein said creatine compound is creatine
phosphate.
18. The method of claim 1, wherein said creatine compound is cyclocreatine.
19. The method of claim 1, wherein said creatine compound is cyclocreatine
phosphate.
20. The method of claim 1, wherein said creatine compound is
homocyclocreatine.
21. The method of claim 1, wherein said subject is a mammal.
22. The method of claim 21, wherein said subject is a human.
23. The method of claim 1, wherein said disease of the nervous system is
selected
from the groups consisting of neuropathies, Alzheimer disease, Parkinson's
disease,
Huntington's disease, amyotropic lateral sclerosis, motor neuron disease,
traumatic nerve
injury, multiple sclerosis, acute disseminated encephalomyelitis, acute
necrotizing
hemorrhagic leukoencephalitis, dysmyelination disease, mitochondrial disease,
migrainous disorder, bacterial infection, fungal infection, stroke, aging,
dementia,
peripheral nervous system diseases and mental disorders such as depression and
schizophrenia.
24. The method of claim 1, wherein said neuroprotective agent is selected from
the
group consisting of approved drugs for the prevention or treatment of
neurodegenerative
diseases, inhibitors of glutamate excitotoxicity, growth factors, nitric oxide
synthase
inhibitors, cyclooxygenase 2 inhibitors, aspirin, ICE inhibitors,
neuroimmunophilis,
N-acetylcystene, antioxidants, lipoic acid, vitamins, cofactors, and CoQ10.

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25. A method for modulating a nervous system disease in a subject, comprising
administering to a subject a therapeutically effective amount of a
combination of a creatine compound and a neuroprotective agent such that a
nervous
system disease is modulated, wherein said creatine compound has the formula:
<IMG>
and pharmaceutically acceptable salts thereof, wherein:
a) Y is selected from the group consisting of: -CO2H, -NHOH, -NO2,
-SO3H, -C(=0)NHSO2J and -P(=O)(OH)(OJ), wherein J is selected from the group
consisting of: hydrogen, C1-C6 straight chain alkyl, C3-C6 branched alkyl, C2-
C6
alkenyl, C3-C6 branched alkenyl, and aryl;
b) A is selected from the group consisting of C, CH, C1-C5alkyl,
C2-C5alkenyl, C2-C5alkynyl, and C1-C5 alkoyl chain, each having 0-2
substituents which
are selected independently from the group consisting of:
1) K, where K is selected from the group consisting of C1-C6
straight alkyl, C2-C6 straight alkenyl, C1-C6 straight alkoyl, C3-C6 branched
alkyl,
C3-C6 branched alkenyl, and C4-C6 branched alkoyl, K having 0-2 substituents
independently selected from the group consisting of bromo, chloro, epoxy and
acetoxy;
2) an aryl group selected from the group consisting of a 1-2 ring
carbocycle and a 1-2 ring heterocycle, wherein the aryl group contains 0-2
substituents
independently selected from the group consisting of -CH2L and -COCH2L where L
is
independently selected from the group consisting of bromo, chloro, epoxy and
acetoxy;
and
3) -NH-M, wherein M is selected from the group consisting of:
hydrogen, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 alkoyl, C3-C4 branched alkyl, C3-
C4
branched alkenyl, and C4 branched alkoyl;
c) X is selected from the group consisting of NR1, CHR1, CR1, O and S,
wherein R1 is selected from the group consisting of:
1) hydrogen;

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2) K where K is selected from the group consisting of C1-C6
straight alkyl, C2-C6 straight alkenyl, C1-C6 straight alkoyl, C3-C6 branched
alkyl,
C3-C6 branched alkenyl, and C4-C6 branched alkoyl, K having 0-2 substituents
independently selected from the group consisting of bromo, chloro, epoxy and
acetoxy;
3) an aryl group selected from the group consisting of a 1-2 ring
carbocycle and a 1-2 ring heterocycle, wherein the aryl group contains 0-2
substituents
independently selected from the group consisting of: -CH2L and -COCH2L where L
is
independently selected from the group consisting of: bromo, chloro, epoxy and
acetoxy;
4) a C5-C9 a-amino-w-methyl-w-adenosylcarboxylic acid attached
via the w-methyl carbon;
5) a C5-C9 a-amino-w-aza-w-methyl-w-adenosylcarboxylic acid
attached via the w-methyl carbon; and
6) a C5-C9 a-amino-w-thia-w-methyl-w-adenosylcarboxylic acid
attached via the w-methyl carbon;
d) Z1 and Z2 are chosen independently from the group consisting of: =0,
-NHR2, -CH2R2, -NR2OH; wherein Z1 and Z2 may not both be =0 and wherein R2 is
selected from the group consisting of:
1) hydrogen;
2) K, where K is selected from the group consisting of C1-C6
straight alkyl; C2-C6 straight alkenyl, C1-C6 straight alkoyl, C3-C6 branched
alkyl,
C3-C6 branched alkenyl, and C4-C6 branched alkoyl, K having 0-2 substituents
independently selected from the group consisting of bromo, chloro, epoxy and
acetoxy;
3) an aryl group selected from the group consisting of a 1-2 ring
carbocycle and a 1-2 ring heterocycle, wherein the aryl group contains 0-2
substituents
independently selected from the group consisting of -CH2L and -COCH2L where L
is
independently selected from the group consisting of bromo, chloro, epoxy and
acetoxy;
4) a C4-C8 a-amino-carboxylic acid attached via the w-carbon;
5) B, wherein B is selected from the group consisting of -CO2H,
-NHOH, -SO3H, -NO2, OP(=O)(OH)(OJ) and -P(=O)(OH)(OJ), wherein J is selected
from the group consisting of: hydrogen, C1-C6 straight alkyl, C3-C6 branched
alkyl,
C2-C6 alkenyl, C3-C6 branched alkenyl, and aryl, wherein B is optionally
connected to
the nitrogen via a linker selected from the group consisting of: C1-C2 alkyl,
C2 alkenyl,
and C1-C2 alkoyl;

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6) -D-E, wherein D is selected from the group consisting of: C1-C3
straight alkyl, C3 branched alkyl, C2-C3 straight alkenyl, C3 branched
alkenyl, C1-C3
straight alkoyl, aryl and aroyl; and E is selected from the group consisting
of:
-(PO3)n NMP, where n is 0-2 and NMP is ribonucleotide monophosphate connected
via
the 5'-phosphate, 3'-phosphate or the aromatic ring of the base; -
[P(=O)(OCH3)(O)]m-Q,
where m is 0-3 and Q is a ribonucleoside connected via the ribose or the
aromatic ring of
the base; -[P(=O)(OH)(CH2)]m-Q, where m is 0-3 and Q is a ribonucleoside
connected
via the ribose or the aromatic ring of the base; and an aryl group containing
0-3
substituents chosen independently from the group consisting of Cl, Br, epoxy,
acetoxy,
-OG, -C(=O)G, and -CO2G, where G is independently selected from the group
consisting of: C1-C6 straight alkyl, C2-C6 straight alkenyl, C1-C6 straight
alkoyl,
C3-C6 branched alkyl, C3-C6 branched alkenyl, C4-C6 branched alkoyl, wherein E
may
be attached to any point to D, and if D is alkyl or alkenyl, D may be
connected at either
or both ends by an amide linkage; and
7) -E, wherein E is selected from the group consisting of
-(PO3)n NMP, where n is 0-2 and NMP is a ribonucleotide monophosphate
connected via
the 5'-phosphate, 3'-phosphate or the aromatic ring of the base; -
[P(=O)(OCH3)(0)]m-Q,
where m is 0-3 and Q is a ribonucleoside connected via the ribose or the
aromatic ring of
the base; -[P(=O)(OH)(CH2)]m-Q, where m is 0-3 and Q is a ribonucleoside
connected
via the ribose or the aromatic ring of the base; and an aryl group containing
0-3
substituents chose independently from the group consisting of: Cl, Br, epoxy,
acetoxy,
-OG, -C(=O)G, and -CO=G, where G is independently selected from the group
consisting of C1-C6 straight alkyl, C2-C6 straight alkenyl, C1-C6 straight
alkoyl, C3-C6
branched alkyl, C3-C6 branched alkenyl, C4-C6 branched alkoyl; and if E is
aryl, E may
be connected by an amide linkage;
e) if R1 and at least one R2 group are present, R1 may be connected by a
single or double bond to an R2 group to form a cycle of 5 to 7 members;
f) if two R2 groups are present, they may be connected by a single or a
double bond to form a cycle of 4 to 7 members; and
g) if R1 is present and Z1 or Z2 is selected from the group consisting of -
NHR2, -CH2R2 and -NR2OH, then R1 may be connected by a single or double bond
to
the carbon or nitrogen of either Z1 or Z2 to form a cycle of 4 to 7 members.

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26. The method of claim 25, wherein said nervous system disease is modulated
by
preventing the occurrence of the disease.
27. The method of claim 25, wherein said neuroprotective agent is a
mitochondrial
cofactor.
28. The method of claim 27, wherein said mitochondrial cofactor is 2,3
dimethoxy-5-methyl-6-decaprenyl benoquinone.
29. The method of claim 25, wherein said neuroprotective agent is an electron
transport chain regulator.
30. The method of claim 29, wherein said electron transport chain regulator is
nicotinamide.
31. The method of claim 25, wherein said neuroprotective agent is a spin trap.
32. The method of claim 31, wherein said spin trap is PBN.
33. The method of claim 25, wherein said neuroprotective agent is a cofactor
for
normal cellular metabolism.
34. The method of claim 33, wherein said cofactor is carnitine.
35. The method of claim 25, wherein said neuroprotective agent is an
antioxidant.
36. The method of claim 35, wherein said antioxidant is vitamin E.
37. The method of claim 25, wherein said neuroprotective agent is a vitamin.
38. The method of claim 37, wherein said vitamin is riboflavin.
39. The method of claim 25, further comprising administering at least one
additional
neuroprotective agent or creatine compound.
40. The method of claim 25, wherein said creatine compound is creatine.
41. The method of claim 25, wherein said subject is a mammal.

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42. The method of claim 25, wherein said subject is a human.
43. The method of claim 25, wherein said disease of the nervous system is
selected
from the group consisting of neuropathies, Alzheimer disease, Parkinson's
disease,
Huntington's disease, amyotropic lateral sclerosis, motor neuron disease,
traumatic nerve
injury, multiple sclerosis,, acute disseminated encephalomyelitis, acute
necrotizing
hemorrhagic leukoencephalitis, dysmyelination disease, mitochondrial disease,
migrainous disorder, bacterial infection, fungal infection, stroke, aging,
dementia,
peripheral nervous system diseases and mental disorders such as depression and
schizophrenia.
44. A pharmaceutical composition for modulating a nervous system disease in a
subject, comprising
a synergistically effective amount of a combination of a creatine compound
having the formula
<IMG>
and pharmaceutically acceptable salts thereof, wherein:
a) Y is selected from the group consisting of: -CO2H, -NHOH, -NO2,
-SO3H, -C(=0)NHSO2J and -P(=O)(OH)(OJ), wherein J is selected from the group
consisting of: hydrogen, C1-C6 straight chain alkyl, C3-C6 branched alkyl, C2-
C6
alkenyl, C3-C6 branched alkenyl, and aryl;
b) A is selected from the group consisting of: C, CH, C1-C5alkyl,
C2-C5alkenyl, C2-C5alkynyl, and C1-C5 alkoyl chain, each having 0-2
substituents which
are selected independently from the group consisting of:
1) K, where K is selected from the group consisting of C1-C6
straight alkyl, C2-C6 straight alkenyl, C1-C6 straight alkoyl, C3-C6 branched
alkyl,
C3-C6 branched alkenyl, and C4-C6 branched alkoyl, K having 0-2 substituents
independently selected from the group consisting of: bromo, chloro, epoxy and
acetoxy;

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2) an aryl group selected from the group consisting of: a 1-2 ring
carbocycle and a 1-2 ring heterocycle, wherein the aryl group contains 0-2
substituents
independently selected from the group consisting of: -CH2L and -COCH2L where L
is
independently selected from the group consisting of bromo, chloro, epoxy and
acetoxy;
and
3) -NH-M, wherein M is selected from the group consisting of:
hydrogen, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 alkoyl, C3-C4 branched alkyl, C3-
C4
branched alkenyl, and C4 branched alkoyl;
c) X is selected from the group consisting of NR1, CHR1, CR1, O and S,
wherein R1 is selected from the group consisting of:
1) hydrogen;
2) K where K is selected from the group consisting of: C1-C6
straight alkyl, C2-C6 straight alkenyl, C1-C6 straight alkoyl, C3-C6 branched
alkyl,
C3-C6 branched alkenyl, and C4-C6 branched alkoyl, K having 0-2 substituents
independently selected from the group consisting of: bromo, chloro; epoxy and
acetoxy;
3) an aryl group selected from the group consisting of a 1-2 ring
carbocycle and a 1-2 ring heterocycle, wherein the aryl group contains 0-2
substituents
independently selected from the group consisting of: -CH2L and -COCH2L where L
is
independently selected from the group consisting of: bromo, chloro, epoxy and
acetoxy;
4) a C5-C9 a-amino-w-methyl-w-adenosylcarboxylic acid attached
via the w-methyl carbon;
5) a C5-C9 a-amino-w-aza-w-methyl-w-adenosylcarboxylic acid
attached via the w-methyl carbon; and
6) a C5-C9 a-amino-w-thia-w-methyl-w-adenosylcarboxylic acid
attached via the w-methyl carbon;
d) Z1 and Z2 are chosen independently from the group consisting of =0,
-NHR2, -CH2R2, -NR2OH; wherein Z1 and Z2 may not both be =0 and wherein R2 is
selected from the group consisting of:
1) hydrogen;
2) K, where K is selected from the group consisting of: C1-C6
straight alkyl; C2-C6 straight alkenyl, C1-C6 straight alkoyl, C3-C6 branched
alkyl,
C3-C6 branched alkenyl, and C4-C6 branched alkoyl, K having 0-2 substituents
independently selected from the group consisting of: bromo, chloro, epoxy and
acetoxy;
3) an aryl group selected from the group consisting of a 1-2 ring
carbocycle and a 1-2 ring heterocycle, wherein the aryl group contains 0-2
substituents

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independently selected from the group consisting of: -CH2L and -COCH2L where L
is
independently selected from the group consisting of: bromo, chloro, epoxy and
acetoxy;
4) a C4-C8 a-amino-carboxylic acid attached via the w-carbon;
5) B, wherein B is selected from the group consisting of -CO2H,
-NHOH, -SO3H, -NO2, OP(=O)(OH)(OJ) and -P(=O)(OH)(OJ), wherein 3 is selected
from the group consisting of: hydrogen, C1-C6 straight alkyl, C3-C6 branched
alkyl,
C2-C6 alkenyl, C3-C6 branched alkenyl, and aryl, wherein B is optionally
connected to
the nitrogen via a linker selected from the group consisting of C1-C2 alkyl,
C2 alkenyl,
and C1-C2 alkoyl;
6) -D-E, wherein D is selected from the group consisting of C1-C3
straight alkyl, C3 branched alkyl, C2-C3 straight alkenyl, C3 branched
alkenyl, C1-C3
straight alkoyl, aryl and aroyl; and E is selected from the group consisting
of
-(PO3)n NMP, where n is 0-2 and NMP is ribonucleotide monophosphate connected
via
the 5'-phosphate, 3'-phosphate or the aromatic ring of the base; -
[P(=O)(OCH3)(0)]m-Q,
where m is 0-3 and Q is a ribonucleoside connected via the ribose or the
aromatic ring of
the base; -[P(=O)(OH)(CH2)]m-Q, where m is 0-3 and Q is a ribonucleoside
connected
via the ribose or the aromatic ring of the base; and an aryl group containing
0-3
substituents chosen independently from the group consisting of Cl, Br, epoxy,
acetoxy,
-OG, -C(=O)G, and -CO2G, where G is independently selected from the group
consisting of C1-C6 straight alkyl, C2-C6 straight alkenyl, C1-C6 straight
alkoyl,
C3-C6 branched alkyl, C3-C6 branched alkenyl, C4-C6 branched alkoyl, wherein E
may
be attached to any point to D, and if D is alkyl or alkenyl, D may be
connected at either
or both ends by an amide linkage; and
7) -E, wherein E is selected from the group consisting of:
(PO3)n NMP, where n is 0-2 and NMP is a ribonucleotide monophosphate connected
via
the 5'-phosphate, 3'-phosphate or the aromatic ring of the base; -
[P(=O)(OCH3)(0)]m-Q,
where m is 0-3 and Q is a ribonucleoside connected via the ribose or the
aromatic ring of
the base; -[P(=O)(OH)(CH2)]m-Q, where m is 0-3 and Q is a ribonucleoside
connected
via the ribose or the aromatic ring of the base; and an aryl group containing
0-3
substituents chose independently from the group consisting of: Cl, Br, epoxy,
acetoxy,
-OG, -C(=O)G, and -CO=G, where G is independently selected from the group
consisting of C1-C6 straight alkyl, C2-C6 straight alkenyl, C1-C6 straight
alkoyl, C3-C6
branched alkyl, C3-C6 branched alkenyl, C4-C6 branched alkoyl; and if E is
aryl, E may
be connected by an amide linkage;

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e) if R1 and at least one R2 group are present, R1 may be connected by a
single or double bond to an R2 group to form a cycle of 5 to 7 members;
f) if two R2 groups are present, they may be connected by a single or a
double bond to form a cycle of 4 to 7 members; and
g) if R1 is present and Z1 or Z2 is selected from the group consisting of
-NHR2, -CH2R2 and -NR2OH, then R1 may be connected by a single or double bond
to
the carbon or nitrogen of either Z1 or Z2 to form a cycle of 4 to 7 members;
and
a neuroprotective agent and a pharmaceutically acceptable carrier.
45. The pharmaceutical composition of claim 44, wherein said creatine compound
is
creatine.
46. The pharmaceutical composition of claim 44, wherein said creatine compound
is
cyclocreatine.
47. A packaged nervous system disease modulator, comprising
a creatine compound having the formula
<IMG>
and pharmaceutically acceptable salts thereof, wherein:
a) Y is selected from the group consisting of: -CO2H, -NHOH, -NO2,
-SO3H, -C(=0)NHS02J and -P(=O)(OH)(OJ), wherein J is selected from the group
consisting of: hydrogen, C1-C6 straight chain alkyl, C3-C6 branched alkyl, C2-
C6
alkenyl, C3-C6 branched alkenyl, and aryl;
b) A is selected from the group consisting of C, CH, C1-C5alkyl,
C2-C5alkenyl, C2-C5alkynyl, and C1-C5 alkoyl chain, each having 0-2
substituents which
are selected independently from the group consisting of:
1) K, where K is selected from the group consisting of: C1-C6
straight alkyl, C2-C6 straight alkenyl, C1-C6 straight alkoyl, C3-C6 branched
alkyl,

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C3-C6 branched alkenyl, and C4-C6 branched alkoyl, K having 0-2 substituents
independently selected from the group consisting of: bromo, chloro, epoxy and
acetoxy;
2) an aryl group selected from the group consisting of: a 1-2 ring
carbocycle and a 1-2 ring heterocycle, wherein the aryl group contains 0-2
substituents
independently selected from the group consisting of: -CH2L and -COCH2L where L
is
independently selected from the group consisting of bromo, chloro, epoxy and
acetoxy;
and
3) -NH-M, wherein M is selected from the group consisting of
hydrogen, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 alkoyl, C3-C4 branched alkyl, C3-
C4
branched alkenyl, and C4 branched alkoyl;
c) X is selected from the group consisting of NR1, CHR1, CR1, O and S,
wherein R1 is selected from the group consisting of:
1) hydrogen;
2) K where K is selected from the group consisting of: C1-C6
straight alkyl, C2-C6 straight alkenyl, C1-C6 straight alkoyl, C3-C6 branched
alkyl,
C3-C6 branched alkenyl, and C4-C6 branched alkoyl, K having 0-2 substituents
independently selected from the group consisting of: bromo, chloro, epoxy and
acetoxy;
3) an aryl group selected from the group consisting of a 1-2 ring
carbocycle and a 1-2 ring heterocycle, wherein the aryl group contains 0-2
substituents
independently selected from the group consisting of -CH2L and -COCH2L where L
is
independently selected from the group consisting of bromo, chloro, epoxy and
acetoxy;
4) a C5-C9 a-amino-w-methyl-w-adenosylcarboxylic acid attached
via the w-methyl carbon;
5) a C5-C9 a-amino-w-aza-w-methyl-w-adenosylcarboxylic acid
attached via the w-methyl carbon; and
6) a C5-C9 a-amino-w-thia-w-methyl-w-adenosylcarboxylic acid
attached via the w-methyl carbon;
d) Z1 and Z2 are chosen independently from the group consisting of: =0,
-NHR2, -CH2R2, -NR2OH; wherein Z1 and Z2 may not both be =0 and wherein R2 is
selected from the group consisting of:
1) hydrogen;
2) K, where K is selected from the group consisting of: C1-C6
straight alkyl; C2-C6 straight alkenyl, C1-C6 straight alkoyl, C3-C6 branched
alkyl,
C3-C6 branched alkenyl, and C4-C6 branched alkoyl, K having 0-2 substituents
independently selected from the group consisting of: bromo, chloro, epoxy and
acetoxy;

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3) an aryl group selected from the group consisting of a 1-2 ring
carbocycle and a 1-2 ring heterocycle, wherein the aryl group contains 0-2
substituents
independently selected from the group consisting of: -CH2L and -COCH2L where L
is
independently selected from the group consisting of: bromo, chloro, epoxy and
acetoxy;
4) a C4-C8 a-amino-carboxylic acid attached via the w-carbon;
5) B, wherein B is selected from the group consisting of: -CO2H,
-NHOH, -SO3H, -NO2, OP(=O)(OH)(OJ) and -P(=O)(OH)(OJ), wherein J is selected
from the group consisting of: hydrogen, C1-C6 straight alkyl, C3-C6 branched
alkyl,
C2-C6 alkenyl, C3-C6 branched alkenyl, and aryl, wherein B is optionally
connected to
the nitrogen via a linker selected from the group consisting of: C1-C2 alkyl,
C2 alkenyl,
and C1-C2 alkoyl;
6) -D-E, wherein D is selected from the group consisting of C1-C3
straight alkyl, C3 branched alkyl, C2-C3 straight alkenyl, C3 branched
alkenyl, C1-C3
straight alkoyl, aryl and aroyl; and E is selected from the group consisting
of:
-(P03)n NMP, where n is 0-2 and NMP is ribonucleotide monophosphate connected
via
the 5'-phosphate, 3'-phosphate or the aromatic ring of the base; -
[P(=O)(OCH3)(0)]m-Q,
where m is 0-3 and Q is a ribonucleoside connected via the ribose or the
aromatic ring of
the base; -[P(=O)(OH)(CH2)]m-Q, where m is 0-3 and Q is a ribonucleoside
connected
via the ribose or the aromatic ring of the base; and an aryl group containing
0-3
substituents chosen independently from the group consisting of: Cl, Br, epoxy,
acetoxy,
-OG, -C(=O)G, and -CO2G, where G is independently selected from the group
consisting of: C1-C6 straight alkyl, C2-C6 straight alkenyl, C1-C6 straight
alkoyl,
C3-C6 branched alkyl, C3-C6 branched alkenyl, C4-C6 branched alkoyl, wherein E
may
be attached to any point to D, and if D is alkyl or alkenyl, D may be
connected at either
or both ends by an amide linkage; and
7) -E, wherein E is selected from the group consisting of
-(P03)n NMP, where n is 0-2 and NMP is a ribonucleotide monophosphate
connected via
the 5'-phosphate, 3'-phosphate or the aromatic ring of the base; -
[P(=O)(OCH3)(0)]m-Q,
where m is 0-3 and Q is a ribonucleoside connected via the ribose or the
aromatic ring of
the base; -[P(=O)(OH)(CH2)]m-Q, where m is 0-3 and Q is a ribonucleoside
connected
via the ribose or the aromatic ring of the base; and an aryl group containing
0-3
substituents chose independently from the group consisting of: Cl, Br, epoxy,
acetoxy,
-OG, -C(=O)G, and -CO=G, where G is independently selected from the group
consisting of: C1-C6 straight alkyl, C2-C6 straight alkenyl, C1-C6 straight
alkoyl, C3-C6

-64-
branched alkyl, C3-C6 branched alkenyl, C4-C6 branched alkoyl; and if E is
aryl, E may
be connected by an amide linkage;
e) if R1 and at least one R2 group are present, R1 may be connected by a
single or double bond to an R2 group to form a cycle of 5 to 7 members;
f) if two R2 groups are present, they may be connected by a single or a
double bond to form a cycle of 4 to 7 members; and
g) if R1 is present and Z1 or Z2 is selected from the group consisting of
-NHR2, -CH2R2 and -NR2OH, then R1 may be connected by a single or double bond
to
the carbon or nitrogen of either Z1 or Z2 to form a cycle of 4 to 7 members;
and
a neuroprotective agent, both packaged with instructions for using an
effective amount
of a combination of the creatine compound and said neuroprotective agent as a
nervous
system disease modulator.
48. A composition comprising a creatine compound having the formula
<IMG>
and physiologically acceptable salts thereof, wherein:
a) Y is selected from the group consisting of -CO2H, -NHOH, -NO2,
-SO3H, -C(=O)NHS02J and -P(=O)(OH)(OJ), wherein J is selected from the group
consisting of hydrogen, C1-C6 straight chain alkyl, C3-C6 branched alkyl, C2-
C6
alkenyl, C3-C6 branched alkenyl, and aryl;
b) A is selected from the group consisting of C, CH, C1-C5alkyl,
C2-C5alkenyl, C2-C5alkynyl, and C1-C5 alkoyl chain, each having 0-2
substituents which
are selected independently from the group consisting of:
1) K, where K is selected from the group consisting of: C1-C6
straight alkyl, C2-C6 straight alkenyl, C1-C6 straight alkoyl, C3-C6 branched
alkyl,
C3-C6 branched alkenyl, and C4-C6 branched alkoyl, K having 0-2 substituents
independently selected from the group consisting of bromo, chloro, epoxy and
acetoxy;

-65-
2) an aryl group selected from the group consisting of: a 1-2 ring
carbocycle and a 1-2 ring heterocycle, wherein the aryl group contains 0-2
substituents
independently selected from the group consisting of: -CH2L and -COCH2L where L
is
independently selected from the group consisting of: bromo, chloro, epoxy and
acetoxy;
and
3) -NH-M, wherein M is selected from the group consisting of:
hydrogen, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 alkoyl, C3-C4 branched alkyl, C3-
C4
branched alkenyl, and C4 branched alkoyl;
c) X is selected from the group consisting of NR1, CHR1, CR1, O and S,
wherein R1 is selected from the group consisting of:
1) hydrogen;
2) K where K is selected from the group consisting of: C1-C6
straight alkyl, C2-C6 straight alkenyl, C1-C6 straight alkoyl, C3-C6 branched
alkyl,
C3-C6 branched alkenyl, and C4-C6 branched alkoyl, K having 0-2 substituents
independently selected from the group consisting of bromo, chloro, epoxy and
acetoxy;
3) an aryl group selected from the group consisting of a 1-2 ring
carbocycle and a 1-2 ring heterocycle, wherein the aryl group contains 0-2
substituents
independently selected from the group consisting of: -CH2L and -COCH2L where L
is
independently selected from the group consisting of bromo, chloro, epoxy and
acetoxy;
4) a C5-C9 a-amino-w-methyl-w-adenosylcarboxylic acid attached
via the w-methyl carbon;
5) a C5-C9 a-amino-w-aza-w-methyl-w-adenosylcarboxylic acid
attached via the w-methyl carbon; and
6) a C5-C9 a-amino-w-thia-w-methyl-w-adenosylcarboxylic acid
attached via the w-methyl carbon;
d) Z1 and Z2 are chosen independently from the group consisting of: =0,
-NHR2, -CH2R2, -NR2OH; wherein Z1 and Z2 may not both be =0 and wherein R2 is
selected from the group consisting of:
1) hydrogen;
2) K, where K is selected from the group consisting of C1-C6
straight alkyl; C2-C6 straight alkenyl, C1-C6 straight alkoyl, C3-C6 branched
alkyl,
C3-C6 branched alkenyl, and C4-C6 branched alkoyl, K having 0-2 substituents
independently selected from the group consisting of bromo, chloro, epoxy and
acetoxy;

-66-
3) an aryl group selected from the group consisting of a 1-2 ring
carbocycle and a 1-2 ring heterocycle, wherein the aryl group contains 0-2
substituents
independently selected from the group consisting of -CH2L and -COCH2L where L
is
independently selected from the group consisting of bromo, chloro, epoxy and
acetoxy;
4) a C4-C8 a-amino-carboxylic acid attached via the w-carbon;
5) B, wherein B is selected from the group consisting of: -CO2H,
-NHOH, -SO3H, -NO2, OP(=O)(OH)(OJ) and -P(=O)(OH)(OJ), wherein J is selected
from the group consisting of hydrogen, C1-C6 straight alkyl, C3-C6 branched
alkyl,
C2-C6 alkenyl, C3-C6 branched alkenyl, and aryl, wherein B is optionally
connected to
the nitrogen via a linker selected from the group consisting of: C1-C2 alkyl,
C2 alkenyl,
and C1-C2 alkoyl;
6) -D-E, wherein D is selected from the group consisting of C1-C3
straight alkyl, C3 branched alkyl, C2-C3 straight alkenyl, C3 branched
alkenyl, C1-C3
straight alkoyl, aryl and aroyl; and E is selected from the group consisting
of:
-(P03)n NMP, where n is 0-2 and NMP is ribonucleotide monophosphate connected
via
the 5'-phosphate, 3'-phosphate or the aromatic ring of the base; -
[P(=O)(OCH3)(0)]m-Q,
where m is 0-3 and Q is a ribonucleoside connected via the ribose or the
aromatic ring of
the base; -[P(=O)(OH)(CH2)]m-Q, where m is 0-3 and Q is a ribonucleoside
connected
via the ribose or the aromatic ring of the base; and an aryl group containing
0-3
substituents chosen independently from the group consisting of: Cl, Br, epoxy,
acetoxy,
-OG, -C(=O)G, and -CO2G, where G is independently selected from the group
consisting of C1-C6 straight alkyl, C2-C6 straight alkenyl, C1-C6 straight
alkoyl,
C3-C6 branched alkyl, C3-C6 branched alkenyl, C4-C6 branched alkoyl, wherein E
may
be attached to any point to D, and if D is alkyl or alkenyl, D may be
connected at either
or both ends by an amide linkage; and
7) -E, wherein E is selected from the group consisting of
-(P03)n NMP, where n is 0-2 and NMP is a ribonucleotide monophosphate
connected via
the 5'-phosphate, 3'-phosphate or the aromatic ring of the base; -
[P(=O)(OCH3)(0)]m-Q,
where m is 0-3 and Q is a ribonucleoside connected via the ribose or the
aromatic ring of
the base; -[P(=O)(OH)(CH2)]m-Q, where m is 0-3 and Q is a ribonucleoside
connected
via the ribose or the aromatic ring of the base; and an aryl group containing
0-3
substituents chose independently from the group consisting of Cl, Br, epoxy,
acetoxy,
-OG, -C(=O)G, and -CO=G, where G is independently selected from the group
consisting of C1-C6 straight alkyl, C2-C6 straight alkenyl, C1-C6 straight
alkoyl, C3-C6

-67-
branched alkyl, C3-C6 branched alkenyl, C4-C6 branched alkoyl; and if E is
aryl, E may
be connected by an amide linkage;
e) if R1 and at least one R2 group are present, R1 may be connected by a
single or double bond to an R2 group to form a cycle of 5 to 7 members;
f) if two R2 groups are present, they may be connected by a single or a
double bond to form a cycle of 4 to 7 members; and
g) if R1 is present and Z1 or Z2 is selected from the group consisting of
NHR2, -CH2R2 and -NR2OH, then R1 may be connected by a single or double bond
to
the carbon or nitrogen of either Z1 or Z2 to form a cycle of 4 to 7 members;
and
a neuroprotective agent.
49. The composition of claim 48, wherein said creatine compound is creatine.
50. The composition of claim 48, wherein said creatine compound is creatine
phosphate.
51. The composition of claim 48, wherein said creatine compound is
cyclocreatine.
52. The composition of claim 48, wherein said creatine compound is
cyclocreatine
phosphate.
53. The composition of claim 48, wherein said neuroprotective agent is
nicotinamide.
54. The composition of claim 48, wherein said neuroprotective agent is CoQ10.
55. The composition of claim 48, wherein said neuroprotective agent is an
anti-oxidant.
56. A composition comprising at least one creatine compound and at least one
ATP
enhancing agent.
57. The composition of claim 56, wherein said composition is a pharmaceutical
composition which further includes a pharmaceutically acceptable carrier and
is for use
in therapy.

-68-
58. The composition of claim 56, wherein said composition is a food or medical
supplement composition.
59. The composition of claim 56, wherein said creatine compound has the
formula
<IMG>
and physiologically acceptable salts thereof, wherein:
a) Y is selected from the group consisting of -CO2H, -NHOH,
-NO2,-SO3H, -C(=0)NHSO2J and -P(=O)(OH)(OJ), wherein J is selected from the
group
consisting of hydrogen, C1-C6 straight chain alkyl, C3-C6 branched alkyl, C2-
C6
alkenyl, C3-C6 branched alkenyl, and aryl;
b) A is selected from the group consisting of: C, CH, C1-C5alkyl,
C2-C5alkenyl, C2-C5alkynyl, and C1-C5 alkoyl chain, each having 0-2
substituents which
are selected independently from the group consisting of:
1) K, where K is selected from the group consisting of: C1-C6
straight alkyl, C2-C6 straight alkenyl, C1-C6 straight alkoyl, C3-C6 branched
alkyl,
C3-C6 branched alkenyl, and C4-C6 branched alkoyl, K having 0-2 substituents
independently selected from the group consisting of: bromo, chloro, epoxy and
acetoxy;
2) an aryl group selected from the group consisting of: a 1-2 ring
carbocycle and a 1-2 ring heterocycle, wherein the aryl group contains 0-2
substituents
independently selected from the group consisting of: -CH2L and -COCH2L where L
is
independently selected from the group consisting of: bromo, chloro, epoxy and
acetoxy;
and
3) -NH-M, wherein M is selected from the group consisting of:
hydrogen, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 alkoyl, C3-C4 branched alkyl, C3-
C4
branched alkenyl, and C4 branched alkoyl;
c) X is selected from the group consisting of NR1, CHR1, CR1, O and S,
wherein R1 is selected from the group consisting of:
1) hydrogen;

-69-
2) K where K is selected from the group consisting of: C1-C6
straight alkyl, C2-C6 straight alkenyl, C1-C6 straight alkoyl, C3-C6 branched
alkyl,
C3-C6 branched alkenyl, and C4-C6 branched alkoyl, K having 0-2 substituents
independently selected from the group consisting of bromo, chloro, epoxy and
acetoxy;
3) an aryl group selected from the group consisting of a 1-2 ring
carbocycle and a 1-2 ring heterocycle, wherein the aryl group contains 0-2
substituents
independently selected from the group consisting of: -CH2L and -COCH2L where L
is
independently selected from the group consisting of bromo, chloro, epoxy and
acetoxy;
4) a C5-C9 a-amino-w-methyl-w-adenosylcarboxylic acid attached
via the w-methyl carbon;
5) a C5-C9 a-amino-w-aza-w-methyl-w-adenosylcarboxylic acid
attached via the w-methyl carbon; and
6) a C5-C9 a-amino-w-thia-w-methyl-w-adenosylcarboxylic acid
attached via the w-methyl carbon;
d) Z1 and Z2 are chosen independently from the group consisting of: =0,
-NHR2, -CH2R2, -NR2OH; wherein Z1 and Z2 may not both be =0 and wherein R2 is
selected from the group consisting of:
1) hydrogen;
2) K, where K is selected from the group consisting of: C1-C6
straight alkyl; C2-C6 straight alkenyl, C1-C6 straight alkoyl, C3-C6 branched
alkyl,
C3-C6 branched alkenyl, and C4-C6 branched alkoyl, K having 0-2 substituents
independently selected from the group consisting of: bromo, chloro, epoxy and
acetoxy;
3) an aryl group selected from the group consisting of a 1-2 ring
carbocycle and a 1-2 ring heterocycle, wherein the aryl group contains 0-2
substituents
independently selected from the group consisting of: -CH2L and -COCH2L where L
is
independently selected from the group consisting of bromo, chloro, epoxy and
acetoxy;
4) a C4-C8 a-amino-carboxylic acid attached via the w-carbon;
5) B, wherein B is selected from the group consisting of
-CO2H, -NHOH, -SO3H, -NO2, OP(=O)(OH)(OJ) and -P(=O)(OH)(OJ), wherein J is
selected
from the group consisting of: hydrogen, C1-C6 straight alkyl, C3-C6 branched
alkyl,
C2-C6 alkenyl, C3-C6 branched alkenyl, and aryl, wherein B is optionally
connected to
the nitrogen via a linker selected from the group consisting of C1-C2 alkyl,
C2 alkenyl,
and C1-C2 alkoyl;

-70-
6) -D-E, wherein D is selected from the group consisting of: C1-C3
straight alkyl, C3 branched alkyl, C2-C3 straight alkenyl, C3 branched
alkenyl, C1-C3
straight alkoyl, aryl and aroyl; and E is selected from the group consisting
of:
-(P0 3)n NMP, where n is 0-2 and NMP is ribonucleotide monophosphate connected
via
the 5'-phosphate, 3'-phosphate or the aromatic ring of the base; -
[P(=O)(OCH3)(0)]m-Q,
where m is 0-3 and Q is a ribonucleoside connected via the ribose or the
aromatic ring of
the base; -[P(=O)(OH)(CH2)]m-Q, where m is 0-3 and Q is a ribonucleoside
connected
via the ribose or the aromatic ring of the base; and an aryl group containing
0-3
substituents chosen independently from the group consisting of Cl, Br, epoxy,
acetoxy,
-OG, -C(=O)G, and -CO2G, where G is independently selected from the group
consisting of: C1-C6 straight alkyl, C2-C6 straight alkenyl, C1 -C6 straight
alkoyl,
C3-C6 branched alkyl, C3-C6 branched alkenyl, C4-C6 branched alkoyl, wherein E
may
be attached to any point to D, and if D is alkyl or alkenyl, D may be
connected at either
or both ends by an amide linkage; and
7) -E, wherein E is selected from the group consisting of
-(P0 3)n NMP, where n is 0-2 and NMP is a ribonucleotide monophosphate
connected via
the 5'-phosphate, 3'-phosphate or the aromatic ring of the base; -
[P(=O)(OCH3)(0)]m-Q,
where m is 0-3 and Q is a ribonucleoside connected via the ribose or the
aromatic ring of
the base; -[P(=O)(OH)(CH2)]m-Q, where m is 0-3 and Q is a ribonucleoside
connected
via the ribose or the aromatic ring of the base; and an aryl group containing
0-3
substituents chose independently from the group consisting of Cl, Br, epoxy,
acetoxy,
-OG, -C(=O)G, and -CO=G, where G is independently selected from the group
consisting of: C 1-C6 straight alkyl, C2-C6 straight alkenyl, C1-C6 straight
alkoyl, C3-C6
branched alkyl, C3-C6 branched alkenyl, C4-C6 branched alkoyl; and if E is
aryl, E may
be connected by an amide linkage;
e) if R1 and at least one R2 group are present, R1 may be connected by a
single or double bond to an R2 group to form a cycle of 5 to 7 members;
f) if two R2 groups are present, they may be connected by a single or a
double bond to form a cycle of 4 to 7 members; and
g) if R1 is present and Z1 or Z2 is selected from the group consisting of
-NHR2, -CH2R2 and -NR2OH, then R1 may be connected by a single or double bond
to
the carbon or nitrogen of either Z1 or Z2 to form a cycle of 4 to 7 members;
and
a neuroprotective agent.

-71-
60. The composition of claim 56, wherein said ATP enhancing agent is selected
from
the group consisting of CoQs, vitamins, spin traps, carnitine, antioxidants,
or
combinations thereof.
61. The composition of claim 56, wherein said ATP enhancing agent is CoQ10.

Description

CA 02327095 2000-10-02
WO 99/51097 PCT/US99/07340
COMPOSITIONS CONTAINING A COMBINATION
OF A CREATIVE COMPOUND AND A SECOND AGENT
Background of the Invention
Creatine is a compound which is naturally occurring and is found in mammalian
brain and other excitable tissues, such as skeletal muscle, retina and heart.
Its
phosphorylated form, creatine phosphate, also is found in the same organs and
is the
product of the creatine kinase reaction utilizing creatine as a substrate.
Creatine and
creatine phosphate can be synthesized relatively easily and are believed to be
non-toxic
to mammals. Kaddurah-Daouk et al. (WO 92/08456 published May 29, 1992 and WO
90/09192, published August 23, 1990; U.S. 5,321,030; and U.S. 5,324,731)
describe
methods of inhibiting the growth, transformation and/or metastasis of
mammalian cells
using related compounds. Examples of compounds described by Kaddurah-Daouk et
al.
include cyclocreatine, b-guandidino propionic acid, homocyclocreatine,
1-carboxymethyl-2-iminohexahydropyrimidine, guanidino acetate and
carbocreatine.
These same inventors have also demonstrated the efficacy of such compounds for
combating viral infections (U.S. 5,321,030). Elgebaly in U.S. Patent 5,091,404
discloses the use of cyclocreatine for restoring functionality in muscle
tissue. Cohn in
PCT publication No. W094/16687 described a method for inhibiting the growth of
several tumors using creatine and related compounds.
Neuroprotective agents can be found in nature and help to maintain an
organisms
ability to function without general distress to the nervous system. Often
times, reduced
levels below what is considered "normal" for these agents, can lead to
diminished
function of the nervous system.
The nervous system is an unresting assembly of cells that continually receives
information, analyzes and perceives it and makes decisions. The principle
cells of the
nervous system are neurons and neuroglial cells. Neurons are the basic
communicating
units of the nervous system and possess dendrites, axons and synapses required
for this
role. Neuroglial cells consist of astrocytes, oligodendrocytes, ependymal
cells, and
microglial cells. Collectively, they are involved in the shelter and
maintenance of
neurons. The functions of astrocytes are incompletely understood but probably
include
the provision of biochemical and physical support and aid in insulation of the
receptive
surfaces of neurons. In addition to their activities in normal brain, they
also react to
CNS injury by glial scar formation. The principle function of the
oligodendrocytes is
the production and maintenance of CNS myelin. They contribute segments of
myelin
sheath to multiple axons.

CA 02327095 2000-10-02
WO 99/51097 _ 2 - PCT/US99/07340
The ependyma cells react to injury mainly by cell loss. Microglial cells
become
activated and assume the shape of a macrophage in response to injury or
destruction of
the brain. These cells can also proliferate and adopt a rod-like form which
could
surround a tiny focus of necrosis or a dead neuron forming a glial nodule.
Microglial
degradation of dead neurons is called neuronophagia.
The creative kinase/creatine phosphate energy system is only one component of
an elaborate energy-generating system found in nervous system cells such as,
for
example, neurons, oligodendrocytes and astrocytes. The components of the
creative
energy system include the enzyme creative kinase, the substrates creative and
creative
phosphate, and the transporter of creative. The reaction catalyzed by
creative kinase is: MgADP ~ PCr + H+ MgATP- + Cr. Some of the functions
associated with this system include efficient regeneration of energy in cells
with
fluctuating and high energy demands, energy transport to different parts of
the cell,
phosphoryl transfer activity, ion transport regulation, and involvement in
signal
transduction pathways.
The creative kinase/phosphocreatine system has been shown to be active in
neurons, astrocytes, oligodendrocytes and Schwann cells. Manos et al., J.
Neurochem.
56:2101-2107 (1991); Molloy et al., J. Neurochem. 59: 1925-1932. The activity
of the
enzyme has been shown to be up-regulated during regeneration and down-
regulated in
degenerative states (see, e.g., Annals Neurology 35(3):331-340 (1994); DeLeon
et al.,
J.Neuruosci. Res. 29:437-448 (1991); Orlovskaia et al. Vestnik Rossiiskoi
Akademii
Meditsinskikh Nauk. 8:34-39 (1992). Burbaeva et al., Shurnal Neuropathologll
Psikhiatrii Imeni S-S-Korsakova 90(7):85-87 (1990); Mitochondria) creative
kinase was
recently found to be the major constituent of pathological inclusions seen in
mitochondria) myopathies. Stadhouders et al., PNAS, 91, pp 5080-5093 (1994).
It is an object of the present invention to provide methods for treatment of
diseases that affect cells of the nervous system that utilize the creative
kinase/phosphocreatine system using compounds which modulate the system.
Summary of the Invention
The present invention is based, at least in part, on the discovery that
certain
combinations of creative compounds and neuroprotective agents, described
infra, can be
used to treat a nervous system disease. Examples of such disease include those
which
there is undesired neuronal activity, characterized by undesirable
demyelinating,
dysmyelinating or degenerative neuronal activity in a mammal. Compositions and
methods of the invention include combinations of creative compounds and
neuroprotective agents. Preferred creative compounds include creative,
creative

CA 02327095 2000-10-02
WO 99/51097 - 3 - PCT/US99/07340_
phosphate, cyclocreatine, cyclocreatine phosphate and beta guanidino propionic
acid.
Preferred neuroprotective agents include: approved drugs for the treatment or
prevention
of neurodegenerative diseases such as Riluzole, Cognex, Aricept, Sinmet,
Sinmet CR,
Permax, Parlodel, Elepryl, Symmetrel, Artane); glutamate excitotoxicity
inhibitors
(such as glutamate uptake and biosynthesis modulation with compounds like
gabapentin
and Riluzole); growth factors like CNTF, BDNF, IGF-1; nitric oxide synthase
inhibitors; cyclo-oxygenase inhibitors such as aspirin; ICE inhibitors;
Neuroimmunophilins; N-acetylcysteine and procysteine; antioxidants, energy
enhancers,
vitamins and cofactors (such as spin traps, CoQlO, carnitine, nicotinamide,
Vit E or D)
and lipoic acid.
The present invention provides methods for modulating a nervous system disease
in a subject by administering to the subject a therapeutically effective
amount ofa
combination of creatine, a creatine phosphate or a creatine analog and a
neuroprotective
agent, such that a nervous system disease is modulated. Additionally, or in
place of the
neuroprotective agent, a creatine compound can be combined with existing
therapeutic
drugs for neurodegenerative diseases.
The present invention also provides methods for modulating a nervous system
disease in a subject by administering to the subject a therapeutically
effective amount of
a combination of a creatine compound and a neuroprotective agent such that a
nervous
system disease is modulated. The creatine compound has the formula:
Zt\
/C-X-A-Y
Z2';
and pharmaceutically acceptable salts thereof, wherein:
a) Y is selected from the group consisting of: -C02H, -NHOH, -N02, -
S03H, -C(=0)NHS02J and -P(=O)(OH)(OJ), wherein J is selected from the group
consisting o~ hydrogen, Cl-C6 straight chain alkyl, C3-C6 branched alkyl, C2-
C6
alkenyl, C3-C6 branched alkenyl, and aryl;

CA 02327095 2000-10-02
WO 99/51097 - 4 - PCT/US99/07340
b) A is selected from the group consisting of: C, CH, Cl-CSalkyl, C2-
Csalkenyl, C2-CSalkynyl, and Cl-CS alkoyl chain, each having 0-2 substituents
which
are selected independently from the group consisting of:
1 ) K, where K is selected from the group consisting of: C 1 -C6
straight alkyl, C2-C6 straight alkenyl, Cl-C6 straight alkoyl, C3-C6 branched
alkyl,
C3-C6 branched alkenyl, and C4-Cg branched alkoyl, K having 0-2 substituents
independently selected from the group consisting of bromo, chloro, epoxy and
acetoxy;
2) an aryl group selected from the group consisting of: a 1-2 ring
carbocycle and a 1-2 ring heterocycle, wherein the aryl group contains 0-2
substituents
independently selected from the group consisting of: -CH2L and -COCH2L where L
is
independently selected from the group consisting of: bromo, chloro, epoxy and
acetoxy;
and
3) -NH-M, wherein M is selected from the group consisting of
hydrogen, Cl-C4 alkyl, C2-C4 alkenyl, Cl-C4 alkoyl, C3-C4 branched alkyl, C3-
C4
branched alkenyl, and C4 branched alkoyl;
c) X is selected from the group consisting of NR1, CHR1, CRl, O and S, wherein
R1 is selected from the group consisting of:
1) hydrogen;
2) K where K is selected from the group consisting of: C1-C6 straight alkyl,
C2-C6 straight alkenyl, Cl-C6 straight alkoyl, C3-C6 branched alkyl, C3-C6
branched
alkenyl, and C4-C6 branched alkoyl, K having 0-2 substituents independently
selected
from the group consisting of: bromo, chloro, epoxy and acetoxy;
3) an aryl group selected from the group consisting of a 1-2 ring
carbocycle and a 1-2 ring heterocycle, wherein the aryl group contains 0-2
substituents
independently selected from the group consisting o~ -CH2L and -COCH2L where L
is
independently selected from the group consisting of: bromo, chloro, epoxy and
acetoxy;
4) a CS-Cg a-amino-w-methyl-w-adenosylcarboxylic acid attached
via the w-methyl carbon;

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S) a CS-Cg a-amino-w-aza-w-methyl-w-adenosylcarboxylic acid
attached via the w-methyl carbon; and
6) a CS-Cg a-amino-w-thia-w-methyl-w-adenosylcarboxylic acid
attached via the w-methyl carbon;
d) Zl and Z2 are chosen independently from the group consisting of =0,
-NHR2, -CH2R2, -NR20H; wherein Z1 and Z2 may not both be =0 and wherein R2 is
selected from the group consisting of:
1 ) hydrogen;
2) K, where K is selected from the group consisting of: Cl-C6
straight alkyl; C2-C6 straight alkenyl, C1-Cg straight alkoyl, C3-C6 branched
alkyl,
C3-C6 branched alkenyl, and C4-C6 branched alkoyl, K having 0-2 substituents
independently selected from the group consisting of: bromo, chloro, epoxy and
acetoxy;
3) an aryl group selected from the group consisting of a 1-2 ring
carbocycle and a 1-2 ring heterocycle, wherein the aryl group contains 0-2
substituents
independently selected from the group consisting of: -CH2L and -COCH2L where L
is
independently selected from the group consisting of: bromo, chloro, epoxy and
acetoxy;
4) a C4-Cg a-amino-carboxylic acid attached via the w-carbon;
5) B, wherein B is selected from the group consisting of -C02H, -
NHOH, -S03H, -N02, OP(=O)(OH)(OJ) and -P(=O)(OH)(OJ), wherein J is selected
from the group consisting of: hydrogen, C 1-C6 straight alkyl, C3-C6 branched
alkyl,
C2-Cg alkenyl, C3-C6 branched alkenyl, and aryl, wherein B is optionally
connected to
the nitrogen via a linker selected from the group consisting of: Cl-C2 alkyl,
C2 alkenyl,
and C1-C2 alkoyl;
6) -D-E, wherein D is selected from the group consisting of C1-C3
straight alkyl, C3 branched alkyl, C2-C3 straight alkenyl, C3 branched
alkenyl, C1-C3
straight alkoyl, aryl and aroyl; and E is selected from the group consisting
of:

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-(p03)nNMP, where n is 0-2 and NMP is ribonucleotide monophosphate connected
via
the S'-phosphate, 3'-phosphate or the aromatic ring of the base; -
[P(=O)(OCH3)(0)]m-Q,
where m is 0-3 and Q is a ribonucleoside connected via the ribose or the
aromatic ring of
the base; -[P(=O)(OH)(CH2)]m-Q, where m is 0-3 and Q is a ribonucleoside
connected
via the ribose or the aromatic ring of the base; and an aryl group containing
0-3
substituents chosen independently from the group consisting of: Cl, Br, epoxy,
acetoxy,
-OG, -C(=O)G, and -C02G, where G is independently selected from the group
consisting of: Cl-C6 straight alkyl, C2-C6 straight alkenyl, C1 -C6 straight
alkoyl,
C3-C6 branched alkyl, C3-C6 branched alkenyl, C4-C6 branched alkoyl, wherein E
may
be attached to any point to D, and if D is alkyl or alkenyl, D may be
connected at either
or both ends by an amide linkage; and
'7) -E, wherein E is selected from the group consisting of -
(p03)nNMP, where n is 0-2 and NMP is a ribonucleotide monophosphate connected
via
the 5'-phosphate, 3'-phosphate or the aromatic ring of the base; -
[P(=O)(OCH3)(0)]m-Q,
where m is 0-3 and Q is a ribonucleoside connected via the ribose or the
aromatic ring of
the base; -[P{=O)(OH)(CH2)]m Q, where m is 0-3 and Q is a ribonucleoside
connected
via the ribose or the aromatic ring of the base; and an aryl group containing
0-3
substituents chose independently from the group consisting of: Cl, Br, epoxy,
acetoxy,
-OG, -C(=O)G, and -CO=G, where G is independently selected from the group
consisting of: C 1-C6 straight alkyl, C2-C6 straight alkenyl, Cl-C6 straight
alkoyl, C3-C6
branched alkyl, C3-C6 branched alkenyl, C4-C6 branched alkoyl; and if E is
aryl, E may
be connected by an amide linkage;
e) if Rl and at least one R2 group are present, Rl may be connected by a
single or double bond to an R2 group to form a cycle of 5 to 7 members;
if two R2 groups are present, they may be connected by a single or a
double bond to form a cycle of 4 to 7 members; and
g) if R1 is present and Z 1 or Z2 is selected from the group consisting of -
NHR2, -CH2R2 and -NR20H, then Rl may be connected by a single or double bond
to
the carbon or nitrogen of either Z1 or Z2 to form a cycle of 4 to 7 members.

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The creatine compound could be combined with a neuroprotective agent selected
from the approved drugs used for the prevention or treatment of
neurodegenerative
diseases).
Neuroprotective agents include: approved drugs for the treatment or prevention
of neurodegenerative diseases such as Riluzole, Cognex, Aricept, Sinmet,
Sinmet CR,
Permax, Parlodel, Elepryl, Symmetrel, Artane); glutamate excitotoxicity
inhibitors
(such as glutamate uptake and biosynthesis modulation with compounds like
gabapentin
and Riluzole); growth factors like CNTF, BDNF, IGF-1; nitric oxide synthase
inhibitors; cyclo-oxygenase inhibitors such as aspirin; ICE inhibitors;
Neuroimmunophilins; N-acetylcysteine and procysteine; antioxidants, energy
enhancers,
vitamins and cofactors (such as spin traps, CoQlO, carnitine, nicotinamide,
Vit E or D)
and lipoic acid.
The present invention further provides pharmaceutical compositions for
modulating a nervous system disease in a subject. The pharmaceutical
compositions
include a synergistically effective amount of a combination of a creatine
compound
having the formula described above, a neuroprotective agent and a
pharmaceutically
acceptable carrier. In preferred embodiments, the creatine compound is
creatine,
creatine phosphate, cyclocreatine or cyclocreatine phosphate and beta
guanidino
propionic acid.
The present invention provides packaged nervous system disease modulators
which include a creatine compound having the formula described above and at
least one
neuroprotective agent. Additionally, or in place of the neuroprotective agent,
a creatine
compound can be combined with existing therapeutic drugs for neurodegenerative
diseases.
Some of the diseases susceptible to treatment with creatine compounds
according
to the present invention include, but are not limited to Alzheimer disease,
Parkinson's
disease, Huntington's disease, motor neuron disease, diabetic and toxic
neuropathies,
traumatic nerve injury, multiple sclerosis, acute disseminated
encephalomyelitis, acute
necrotizing hemorrhagic leukoencephalitis, diseases of dysmyelination,
mitochondria)
diseases, fungal and bacterial infections, migrainous disorders, stroke,
aging, dementia,
and mental disorders such as depression and schizophrenia.
The present invention also provides compositions of creatine compounds,
including the formula described above, and neuroprotective agents. Preferred
creatine
compounds include creatine, creatine phosphate, cyclocreatine or cyclocreatine
phosphate and beta guanidino propionic acid. Preferred neuroprotective agents
include
approved drugs for the treatment or prevention of neurodegenerative diseases
such as
Riluzole, Cognex, Aricept, Sinmet, Sinmet CR, Permax, Parlodel, Elepryl,
Symmetrel,
Artane); glutamate excitotoxicity inhibitors (such as glutamate uptake and
biosynthesis

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modulation with compounds like gabapentin and Riluzole); growth factors like
CNTF,
BDNF, IGF-1; nitric oxide synthase inhibitors; cyclo-oxygenase inhibitors such
as
aspirin; ICE inhibitors; Neuroimmunophilins; N-acetylcysteine and procysteine;
antioxidants, energy enhancers, vitamins and cofactors (such as spin traps,
CoQlO,
camitine, nicotinamide, Vit E or D) and lipoic acid.
The present invention further provides compositions of creative compounds,
including the formula described above, and neuroprotective agents developed as
a
neutritional supplement, medical food or drug.form. Preferred creative
compounds
include creative, creative phosphate, cyclocreatine or cyclocreatine phosphate
or beta
guanidino propionic acid. Preferred neuroprotective agents include: approved
drugs for
the treatment or prevention of neurodegenerative diseases such as Riluzole,
Cognex,
Aricept, Sinmet, Sinmet CR, Permax, Parlodel, Elepryl, Symmetrel, Artane);
glutamate
excitotoxicity inhibitors (such as glutamate uptake and biosynthesis
modulation with
compounds like gabapentin and Riluzole); growth factors like CNTF, BDNF, IGF-
1;
nitric oxide synthase inhibitors; cyclo-oxygenase inhibitors such as aspirin;
ICE
inhibitors; Neuroimmunophilins; N-acetylcysteine and procysteine;
antioxidants, energy
enhancers, vitamins and cofactors (such as spin traps, CoQlO, carnitine,
nicotinamide,
Vit E or D) and lipoic acid.
Brief Description of the Figures
Figure 1 is a graph illustrating the effect of creative and cyclocreatine on
lesion
volumes in mice using the malonate model.
Figure 2 is a graph illustrating the dose-response effects of creative and
cyclocreatine on lesion volumes in mice using the malonate model.
Figure 3 is a graph illustrating the effect of creative on lesion volumes in
mice
using the 3-NP model.
Figure 4 is a graph illustrating the effect of creative and cyclocreatine on
levels
of dopamine, HVA, and DOPAC in mice using the MPTP model.
Figure 5 is a graph illustrating the dose-response effects of creative and
cyclocreatine on levels of dopamine, HVA and DOPAC in mice using the MPTP
model.
Figure 6 is a graph illustrating the effect of creative in slowing the rate of
motoneural degeneration of FALS mice.
Figure 7 is a graph illustrating the effect of creative on improving the
survival
times of FALS mice.
Detailed Description
The features and other details of the invention will now be more particularly
described and pointed out in the claims. It will be understood that the
particular

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embodiments of the invention are shown by way of illustration and not as
limitations of
the invention. The principle features of this invention can be employed in
various
embodiments without departing from the scope of the invention.
The methods of the present invention generally comprise administering to an
individual afflicted with a disease of the nervous system a therapeutically
effective
amount of a creatine compound or compounds in combination with a
neuroprotective
agent or agents which modulate one or more of the structural or functional
components
of the creatine kinase/phosphocreatine system sufficient to prevent, reduce or
ameliorate
symptoms of the disease. Components of the system which can be modulated
include
the enzyme creative kinase, the substrates creative and creative phosphate,
and the
transporter of creative. As used herein, the term "modulate" means to change,
affect or
interfere with the functions of the creative kinase system.
The present invention is based, at least in part, on the discovery that
certain
combinations of creative compounds and neuroprotective agents, described
infra, can be
used to treat a nervous system disease. Examples of such diseases include
those which
there is undesired neuronal activity, characterized by undesirable
demyelinating,
dysmyelinating or degenerative neuronal activity in a mammal. Compositions and
methods of the invention include combinations of creative compounds and
neuronal
modulatory agents. Preferred creative compounds include creative, creative
phosphate,
cyclocreatine and cyclocreatine phosphate or beta guanidino propionic acid.
Preferred
neuroprotective agents include: approved drugs for the treatment or prevention
of
neurodegenerative diseases such as Riluzole, Cognex, Aricept, Sinmet, Sinmet
CR,
Permax, Parlodel, Elepryl, Symmetrel, Artane); glutamate excitotoxicity
inhibitors
(such as glutamate uptake and biosynthesis modulation with compounds like
gabapentin
and Riluzole); growth factors like CNTF, BDNF, IGF-1; nitric oxide synthase
inhibitors; cyclo-oxygenase inhibitors such as aspirin; ICE inhibitors;
Neuroimmunophilins; N-acetylcysteine and procysteine; antioxidants, energy
enhancers,
vitamins and cofactors (such as spin traps, CoQ 10, carnitine, nicotinamide,
Vit E or D)
and lipoic acid. The creative compounds could be combined with different
neuroprotective agents and administered together or sequentially.
The present invention pertains to methods for modulating a nervous system
disease in a subject by administering to the subject a therapeutically
effective amount of
a combination of creative, a creative phosphate or a creative analog and a
neuroprotective agent, such that a nervous system disease is modulated.
Additionally, or
in place of the neuroprotective agent, a creative compound can be combined
with
existing therapeutic drugs for neurodegenerative diseases.

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Creative compounds which are particularly effective for this purpose include
creative, creative phosphate, and analogs thereof which are described in
detail below.
The term "creative compounds" will be used herein to include creative,
creative
phosphate, and compounds which are structurally similar to creative or
creative
phosphate, and analogs of creative and creative phosphate: The term "creative
compounds" also includes compounds which "mimic" the activity of creative,
creative
phosphate or creative analogs, i.e., compounds which inhibit or modulate the
creative
kinase system. The term creative compound is also intended to include
pharmaceutically
acceptable or physiologically acceptable salts of the compounds. Creative
compounds
have previously been described in copending application Ser. No. 07/061,677
entitled
Methods of Treating Body Parts Susceptible to Ischemia Using Creative Analogs,
filed
May 14, 1993; copending application Ser. No. 08/009,638 entitled Creative
Phosphate,
Creative Phosphate Analogs and Uses Therefor, filed on Jan. 27, 1993;
copending
application Ser. No. 07/812,561 entitled Creative Analogs Having Antiviral
Activity,
filed Dec. 20, 1991; and copending application Ser. No. 07/610,418 entitled
Method of
Inhibiting transformation of Cells in Which Purine Metabolic Enzyme Activity
is
Elevated, filed Nov. 7, 1990. The entire contents of each of the copending
applications
are herein expressly incorporated by reference, along with their published
foreign
counterparts; and all of the creative compounds along with their methods of
synthesis
and discussed in the aforementioned applications are intended to be part of
this invention
unless specifically stated otherwise.
The term "mimics" is intended to include compounds which may not be
structurally similar to creative but mimic the therapeutic activity of
creative, creative
phosphate or structurally similar compounds. The term "inhibitors of creative
kinase
system" are compounds which inhibit the activity of the creative kinase
enzyme,
molecules that inhibit the creative transporter or molecules that inhibit the
binding of the
enzyme to other structural proteins, enzymes or lipids. The term "modulators
of the
creative kinase system" are compounds which modulate the activity of the
enzyme, or
the activity of the transporter of creative or the ability of other proteins
or enzymes or
lipids to interact with the system. The term "creative analog" is intended to
include
compounds which are structurally similar to creative or creative phosphate,
compounds
which are art-recognized as being analogs of creative or creative phosphate,
and/or
compounds which share the same or similar function as creative or creative
phosphate.
The language "modulating a nervous system disease" or "modulating a disease of
the nervous system" is intended to include prevention of the disease,
amelioration and/or
arrest of a preexisting disease, or the elimination of a preexisting disease.
The
combinations of creative analogs and neuroprotective agents described herein
have both
curative and prophylactic effects on disease development and progression.

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The language "therapeutically effective amount" is intended to include the
amount of a combination of a creatine compound and neuroprotective agent
sufficient to
prevent onset of diseases of the nervous system or significantly reduce
progression of
such diseases in the subject being treated. A therapeutically effective amount
can be
determined on an individual basis and will be based, at least in part, on
consideration of
the severity of the symptoms to be treated and the activity of the specific
analog selected
if an analog is being used. Further, the effective amounts of the creatine
compounds)
and neuroprotective agents) may vary according to the age, sex and weight of
the
subject being treated. Thus, a therapeutically effective amount of the
combinations can
be determined by one of ordinary skill in the art employing such factors as
described
above using no more than routine experimentation in clinical management.
The present invention also pertains to methods for modulating a nervous system
disease in a subject by administering to the subject a therapeutically
effective amount of
a combination of a creatine compound and a neuroprotective agent such that a
nervous
system disease is modulated. The creatine compound has the formula:
Zt\C-X-A-Y
/,
and pharmaceutically acceptable salts thereof, wherein:
a) Y is selected from the group consisting of -C02H, -NHOH, -N02, -
S03H, -C(=0)NHS02J and -P(=O)(OH)(OJ), wherein J is selected from the group
consisting of hydrogen, Cl-C6 straight chain alkyl, C3-C6 branched alkyl, C2-
C6
alkenyl, C3-C6 branched alkenyl, and aryl;
b) A is selected from the group consisting of: C, CH, Cl-CSalkyl, C2-
Csalkenyl, C2-CSalkynyl, and Cl-CS alkoyl chain, each having 0-2 substituents
which
are selected independently from the group consisting of:
1) K, where K is selected from the group consisting of C1 -C6
straight alkyl, C2-C6 straight alkenyl, Cl-C6 straight alkoyl, C3-C6 branched
alkyl,
C3-C6 branched alkenyl, and C4-C6 branched alkoyl, K having 0-2 substituents
independently selected from the group consisting of bromo, chloro, epoxy and
acetoxy;

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2) an aryl group selected from the group consisting of: a 1-2 ring
carbocycle and a 1-2 ring heterocycle, wherein the aryl group contains 0-2
substituents
independently selected from the group consisting o~ -CH2L and -COCH2L where L
is
independently selected from the group consisting of bromo, chloro, epoxy and
acetoxy;
and
3) -NH-M, wherein M is selected from the group consisting of
hydrogen, Cl-C4 alkyl, C2-C4 alkenyl, Cl-C4 alkoyl, C3-C4 branched alkyl, C3-
C4
branched alkenyl, and C4 branched alkoyl;
c) X is selected from the group consisting of NR1, CHRl, CR1, O and S,
wherein R1 is selected from the group consisting of:
1) hydrogen;
2) K where K is selected from the group consisting of C1-C6
straight alkyl, C2-C6 straight alkenyl, Cl-C6 straight alkoyl, C3-C6 branched
alkyl,
C3-C6 branched alkenyl, and C4-C6 branched alkoyl, K having 0-2 substituents
independently selected from the group consisting of: bromo, chloro, epoxy and
acetoxy;
3) an aryl group selected from the group consisting of a 1-2 ring
carbocycle and a 1-2 ring heterocycle, wherein the aryl group contains 0-2
substituents
independently selected from the group consisting of -CH2L and -COCH2L where L
is
independently selected from the group consisting of bromo, chloro, epoxy and
acetoxy;
4) a CS-Cg a-amino-w-methyl-w-adenosylcarboxylic acid attached
via the w-methyl carbon;
5) a CS-Cg a-amino-w-aza-w-methyl-w-adenosylcarboxylic acid
attached via the w-methyl carbon; and
6) a CS-Cg a-amino-w-thia-w-methyl-w-adenosylcarboxylic acid
attached via the w-methyl carbon;

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d) Zl and Z2 are chosen independently from the group consisting of =0,
-NHR2, -CH2R2, -NR20H; wherein Z 1 and Z2 may not both be =0 and wherein R2 is
selected from the group consisting of:
1) hydrogen;
2) K, where K is selected from the group consisting of C1-C6
straight alkyl; C2-C6 straight alkenyl, C 1-C6 straight alkoyl, C3-C6 branched
alkyl,
C3-C6 branched alkenyl, and C4-C6 branched alkoyl, K having 0-2 substituents
independently selected from the group consisting of bromo, chloro, epoxy and
acetoxy;
3) an aryl group selected from the group consisting of a 1-2 ring
carbocycle and a 1-2 ring heterocycle, wherein the aryl group contains 0-2
substituents
independently selected from the group consisting of -CH2L and -COCH2L where L
is
independently selected from the group consisting of bromo, chloro, epoxy and
acetoxy;
4) a C4-Cg a-amino-carboxylic acid attached via the w-carbon;
5) B, wherein B is selected from the group consisting of -C02H, -
NHOH, -S03H, -N02, OP(=O)(OH)(OJ) and -P(=O)(OH)(OJ), wherein J is selected
from the group consisting of hydrogen, C1-C6 straight alkyl, C3-C6 branched
alkyl,
C2-C6 alkenyl, C3-C6 branched alkenyl, and aryl, wherein B is optionally
connected to
the nitrogen via a linker selected from the group consisting of: Cl-C2 alkyl,
C2 alkenyl,
and C 1-C2 alkoyl;
6) -D-E, wherein D is selected from the group consisting of C1-C3
straight alkyl, C3 branched alkyl, C2-C3 straight alkenyl, C3 branched
alkenyl, C1-C3
straight alkoyl, aryl and aroyl; and E is selected from the group consisting
of
-(P03)nNMP, where n is 0-2 and NMP is ribonucleotide monophosphate connected
via
the 5'-phosphate, 3'-phosphate or the aromatic ring of the base; -
[P(=O)(OCH3)(0)]m-Q,
where m is 0-3 and Q is a ribonucleoside connected via the ribose or the
aromatic ring of
the base; -[P(=O)(OH)(CH2)]m-Q, where m is 0-3 and Q is a ribonucleoside
connected
via the ribose or the aromatic ring of the base; and an aryl group containing
0-3
substituents chosen independently from the group consisting of: Cl, Br, epoxy,
acetoxy,

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-OG, -C(=O)G, and -C02G, where G is independently selected from the group
consisting of: Cl-C6 straight alkyl, C2-C6 straight alkenyl, C 1 -C6 straight
alkoyl,
C3-C6 branched alkyl, C3-C6 branched alkenyl, C4-C6 branched alkoyl, wherein E
may
be attached to any point to D, and if D is alkyl or alkenyl, D may be
connected at either
or both ends by an amide linkage; and
7) -E, wherein E is selected from the group consisting of -
(P03)nNMP, where n is 0-2 and NMP is a ribonucleotide monophosphate connected
via
the 5'-phosphate, 3'-phosphate or the aromatic ring of the base; -
[P(=O)(OCH3)(0)]m-Q,
where m is 0-3 and Q is a ribonucleoside connected via the ribose or the
aromatic ring of
the base; -[P(=O)(OH)(CH2)]m-Q, where m is 0-3 and Q is a ribonucleoside
connected
via the ribose or the aromatic ring of the base; and an aryl group containing
0-3
substituents chose independently from the group consisting of: Cl, Br, epoxy,
acetoxy,
-OG, -C(=O)G, and -CO=G, where G is independently selected from the group
consisting of C 1-C6 straight alkyl, C2-C6 straight alkenyl, Cl-C6 straight
alkoyl, C3-C6
branched alkyl, C3-C6 branched alkenyl, C4-C6 branched alkoyl; and,if E is
aryl, E may
be connected by an amide linkage;
e) if Rl and at least one R2 group are present, Rl may be connected by a
single or double bond to an R2 group to foam a cycle of 5 to 7 members;
f) if two R2 groups are present, they may be connected by a single or a
double bond to form a cycle of 4 to 7 members; and
g) if Rl is present and Z1 or Z2 is selected from the group consisting of -
NHR2, -CH2R2 and -NR20H, then Rl may be connected by a single or double bond
to
the carbon or nitrogen of either Z 1 or Z2 to form a cycle of 4 to 7 members.
Additionally, or in place of the neuroprotective agent, a creatine compound
can
be combined with existing therapeutic drugs for neurodegenerative diseases.
The term "neuroprotective agent" is intended to include those compositions
which prevent depletion of ATP prevent glutamate excitotoxicity or prevent
production
of free radicals or other agents which interfere with, destroy, or diminish
nervous system
activity. Representative neuroprotective agents include approved drugs for the
treatment
or prevention of neurodegenerative diseases such as Riluzole, Cognex, Aricept,
Sinmet,
Sinmet CR, Permax, Parlodel, Elepryl, Symmetrel, Artane); glutamate
excitotoxicity

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inhibitors (such as glutamate uptake and biosynthesis modulation with
compounds like
gabapentin and Riluzole); growth factors like CNTF, BDNF, IGF-l; nitric oxide
synthase inhibitors; cyclo-oxygenase inhibitors such as aspirin; ICE
inhibitors;
Neuroimmunophilins; N-acetylcysteine and procysteine; antioxidants, energy
enhancers,
vitamins and cofactors (such as spin traps, CoQlO, carnitine, nicotinamide,
Vit E or D)
and lipoic acid.
The present invention further pertains to pharmaceutical compositions for
modulating a nervous system disease in a subject. The pharmaceutical
compositions
include an effective amount, e.g. synergistically effective amount, of a
combination of a
creatine compound having the formula described above, a neuroprotective agent
and a
pharmaceutically acceptable carrier. In preferred embodiments, the creatine
compound
is creatine, creatine phosphate, cyclocreatine or cyclocreatine phosphate beta
guanidino
propionic acid.
The present invention also pertains to packaged nervous system disease
modulators which include a creatine compound having the formula described
above and
at least one neuroprotective agent. Additionally, or in place of the
neuroprotective
agent, a creatine compound can be combined with existing therapeutic drugs for
neurodegenerative diseases.
The language "pharmaceutically acceptable carrier" is intended to include
substances capable of being coadministered with the creatine compounds) and
neuroprotective agents) and which allows the active ingredients to perform
their
intended function of preventing, ameliorating, arresting, or eliminating a
diseases) of
the nervous system. Examples of such carriers include agents to enhance
creatine
compound uptake such as sugars, solvents, dispersion media, adjuvants, delay
agents
and the like. The use of such media and agents for pharmaceutically active
substances is
well known in the art. Any conventional media and agent compatible with the
creatine
compound may be used within this invention.
The term "pharmaceutically acceptable salt" is intended to include art-
recognized
pharmaceutically acceptable salts. Typically these salts are capable of being
hydrolyzed
under physiological conditions. Examples of such salts include sodium,
potassium and
hemisulfate. The term further is intended to include lower hydrocarbon groups
capable
of being hydrolyzed under physiological conditions, i.e. groups which esterify
the
carboxyl moiety, e.g. methyl, ethyl and propyl.
The term "subject" is intended to include living organisms susceptible to
having
diseases of the nervous system, e.g. mammals. Examples of subjects include
humans,
dogs, cats, horses, cows, goats, rats and mice. The term "subject" further is
intended to
include transgenic species.

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The present invention pertains to compositions of creatine compounds,
including
the formula described above, and neuroprotective agents improved nervous
system
function. Preferred creatine compounds include creatine, creatine phosphate,
cyclocreatine or cyclocreatine phosphate beta guanidino propionic acid.
Preferred
neuroprotective agents include: approved drugs for the treatment or prevention
of
neurodegenerative diseases such as Riluzole, Cognex, Aricept, Sinmet, Sinmet
CR,
Permax, Parlodel, Elepryl, Symmetrel, Artane); glutamate excitotoxicity
inhibitors
(such as glutamate uptake and biosynthesis modulation with compounds like
gabapentin
and Riluzole); growth factors like CNTF, BDNF, IGF-1; nitric oxide synthase
inhibitors; cyclo-oxygenase inhibitors such as aspirin; ICE inhibitors;
Neuroimmunophilins; N-acetylcysteine and procysteine; antioxidants, energy
enhancers,
vitamins and cofactors (such as spin traps, CoQlO, carnitine, nicotinamide,
Vit E or D)
and lipoic acid.
These compositions of creatine compounds and neuroprotective agents can be
used as
dietary food supplements or medical foods to improve nervous system activities
and
associated functions. When used as a dietary food supplement or a medical
food, these
compositions are included as additives to enhance the ability of the food to
protect ,
alleviate, andlor enhance the nervous system against nervous system disease
states.
The language "diseases of the nervous system" or "nervous system disease" is
intended to include diseases of the nervous system whose onset, amelioration,
arrest, or
elimination is effectuated by the creatine compounds described herein.
Examples of
types of diseases of the nervous system include demyelinating, dysmyelinating
and
degenerative diseases. Examples of locations on or within the subject where
the diseases
may originate and/or reside include both central and peripheral loci. As the
term
"disease" is used herein, it is understood to exclude, and only encompass
maladies
distinct from, neoplastic pathologies and tumors of the nervous system,
inschemic injury
and viral infections of the nervous system. Examples of types of diseases
suitable for
treatment with the methods and compounds of the instant invention are
discussed in
detail below.
Diseases of the Nervous System
Diseases of the nervous system fall into two general categories: (a)
pathologic
processes such as infections, trauma and neoplasma found in both the nervous
system
and other organs; and, (b) diseases unique to the nervous system which include
diseases
of myelin and systemic degeneration of neurons.

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Of particular concern to neurologists and other nervous system practitioners
are
diseases of (a) demyelination which can develop due to infection, autoimmune
antibodies, and macrophage destruction; and, (b) dysmyelination which result
from
structural defects in myelin.
Diseases of neurons can be the result of (a) aberrant migration of neurons
during
embryogenesis and early stage formation; or (b) degenerative diseases
resulting from a
decrease in neuronal survival, such as occurs in, for example, Alzheimer's
disease,
Parkinson's disease, Huntington's disease, motor neuron disease, ischemia-
related
disease and stroke, and diabetic neuropathy.
Demyelinating Diseases:
Primary demyelination is a loss of myelin sheaths with relative preservation
of
the demyelinated axons. It results either from damage to the oligodendroglia
which
make the myelin or from a direct, usually immunologic or toxic attack on the
myelin
itself. Secondary demyelination, in contrast, occurs following axonal
degeneration. The
demyelinating diseases are a group of CNS conditions characterized by
extensive
primary demyelination. They include multiple sclerosis and its variants and
perivenous
encephalitis. There are several other diseases in which the principal
pathologic change is
primary demyelination, but which are usually conveniently classified in other
categories
such as inborn errors of metabolism, the leukodystrophies, viral disease
(progressive
multifocal leukoencephalopathy PM), as well as several other rare disorders of
unclear
etiology.
Multiple Sclerosis~MS)
Multiple sclerosis is a disease of the central nervous system (CNS) that has a
peak onset of 30-40 years. It affects all parts of the CNS and causes
disability related to
visual, sensory, motor, and cerebellar systems. The disease manifestations can
be mild
and intermittent or progressive and devastating.
The pathogenesis is due to an autoimmune attack on CNS myelin. The
treatments available are symptomatic treating spasticity, fatigue, bladder
dysfunction,
and spasms. Other treatments are directed towards stopping the immunologic
attack on
myelin. These consist of corticosteroids such as prednisone and
methylprednisolone,
general immunosuppressants such as cyclophosphamide and azathioprine, and
immunomodulating agents such as beta-interferon. No treatments are available
to
preserve myelin or make it resistant to attacks.

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Acute Disseminated Encephalomyelitis
Acute Disseminated Encephalomyelitis usually occurs following a viral
infection
and is thought to be due to an autoimmune reaction against CNS myelin,
resulting in
paralysis, lethargy, and coma. It differs from MS by being a monophasic
disease
whereas MS is characterized by recurrence and chronicity. Treatment consists
of
administration of steroids.
Acute Necrotizing Hemorrhagic Leukoencephalitis
This is a rare disease that is generally fatal. It is also thought to be
mediated by
autoimmune attack on CNS myelin that is triggered by a viral infection.
Neurologic
symptoms develop abruptly with headache, paralysis and coma. Death usually
follows
within several days. Treatment is supportive.
Leukodystrophies
These are diseases of the white matter resulting from an error in the myelin
metabolism that leads to impaired myelin formation. They are thought of as
dysmyelinating diseases, and can become manifest at an early age.
Metachromatic Leukodystrophy: an autosomal recessive (inherited) disorder due
to deficiency of the enzyme arylsulfatase A leading to accumulation of lipids.
There is
demyelination in the CNS and peripheral nervous system leading to progressive
weakness and spasticity.
Krabbe's disease: Also inherited as autosomal recessive and due to deficiency
of
another enzyme: galactocerebroside beta-galactosidase.
Adrenoleukodystrophy and adrenomyeloneuropathy: affect the adrenal glad in
addition to the nervous system.
No treatment is available to any of the leukodystrophies except for supportive
treatment
Degenerative Diseases:
There is no good etiology or pathophysiology known for these diseases, and no
compelling reason to assume that they all have a similar etiology. Diseases
under this
category have general similarities. They are diseases of neurons that tend to
result in
selective impairment, affecting one or more functional systems of neurons
while leaving
others intact.
Parkinson's Disease:
Parkinson's disease is due to loss of dopaminergic neurones in the substantia
nigra of the brain. It is manifested by slowed voluntary movements, rigidity,

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expressionless face and stooped posture. Several drugs are available to
increase
dopaminergic function such as levodopa, carbidopa, bromocriptine, pergolide,
or
decrease cholinergic function such as benztropine, and amantadine. Selegiline
is a new
treatment designed to protect the remaining dopaminergic neurons.
Spinocerebellar Degenerations
This is a group of degenerative diseases that affects in varying degrees the
basal
ganglia, brain stem, cerebellum, spinal cord, and peripheral nerves. Patients
present
symptoms of Parkinsonism, ataxia, spasticity, and motor and sensory deficits
reflecting
damage to different anatomic areas and/or neuronal systems in the CNS.
Degenerative Disease Affecting Motor Neurons
Included in this category are diseases such as amyotrophic lateral sclerosis
(ALS), and spinal muscular atrophy. They are characterized by degeneration of
motor
neurones in the CNS leading to progressive weakness, muscle atrophy, and death
caused
by respiratory failure. Treatments are only symptomatic, there are no
available
treatments to slow down or stop the disease.
Alzheimer Disease (AD):
This disease is characterized clinically by slow erosion of mental function,
culminating in profound dementia. The diagnostic pathologic hallmark of AD is
the
presence of large numbers of senile plagues and neurofibrillary tangles in the
brain
especially in neocortex and hippocampus. Loss of specific neuron populations
in these
brain regions and in several subcortical nuclei correlates with depletion in
certain
neurotransmitters including acetylcholine. The etiology of AD is still
unknown. To date
a lot of research has focused on the composition and genesis of the B/A4
amyloid
component of senile plagues. Alzheimer's disease is characterized clinically
by the slow
erosion of intellectual function with the development of profound dementia.
There are
no treatments that slow the progression.
Huntington Disease (HD):
HD is an autosomal dominant disorder of midlife onset, characterized
clinically
by movement disorder, personality changes, and dementia often leading to death
in
15-20 years. The neuropathologic changes in the brain are centered in the
basal ganglia.
Loss of a class of projection neurons, called "spiny cells" because of their
prominent
dendritic spinous processes, is typical. This class of cells contains gamma-
aminobutyric
acid (GABA), substance P, and opioid peptides. Linkage studies have localized
the gene
for HD to the most distal band of the short arm of chromosome 4. No treatments
are

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available that have been shown to retard progression of the disease.
Experimental
studies showing a similarity between neurons that are susceptible to N-methyl
d-aspartate (NMDA) agonists and those that disappear in HD has led to
encouraging
speculation that NMDA antagonists might prove beneficial. Some recent studies
suggest
S that a defect in brain energy metabolism might occur in HD and enhance
neuronal
vulnerability to excitotoxic stress.
Mitochondria) Encephalomyopathies:
Mitochondria) encephalomyopathies are a heterogenous group of disorders
affecting mitochondria) metabolism. These deficits could involve substrate
transport,
substrate utilization, defects of the Krebs Cycle, defects of the respiratory
chain, and
defects of oxidation/phosphorylation coupling. Pure myopathies vary
considerably with
respect to age at onset, course (rapidly progressive, static, or even
reversible), and
distribution of weakness (generalized with respiratory failure, proximal more
than distal
facioscapulohumeral, orbicularis and extraocular muscles with ptosis and
progressive
external ophthalmoplegia). Patients with mitochondria) myopathies complain of
exercise intolerance and premature fatigue.
Peripheral Nervous System Disorders
The peripheral nervous system (PNS) consists of the motor and sensory
components of the cranial and spinal nerves, the autonomic nervous system with
its
sympathetic and parasympathetic divisions, and the peripheral ganglia. It is
the conduit
for sensory information to the CNS and effector signals to the peripheral
organs such as
muscle. Contrary to the brain, which has no ability to regenerate, the
pathologic
reactions of the PNS include both degeneration and regeneration. There are
three basic
pathological processes: Wallerian degeneration, axonal degeneration and
segmental
demyelination that could take place.
Some of the neuropathic syndromes include:
Acute ascending motor paralysis with variable sensory disturbance; examples
being acute demyelinating neuropathics, infectious mononucleosis with
polyneuritis,
hepatitis and polyneuritis, toxic polyneuropathies.
Subacute sensorimotor polyneuropathy; examples of acquired axonal
neurophathics include paraproteinemias, uremia diabetes, amyloidosis,
connective tissue
diseases and leprosy. Examples of inherited diseases include mostly chronic
demyelination with hypertrophic changes, such as peroneal muscular atrophy,
hypertrophic polyneuropathy and Refsum's diseases.

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Chronic relapsing polyneuropathy; such as idiopathic polyneuritis porphyria,
Beriberi and intoxications.
Mono or multiple neuropathy; such as pressure palsies, traumatic palsies,
serum
neuritis, zoster and leprosy.
Aging:
During the process of aging increased oxidative damage and impaired
mitochondrial
functions contribute to neuronal cell death. Mitochondria are deeply involved
in the
production of reactive oxygen species and are themselves highly susceptible to
oxidative
stress which results in apoptotic cell death. Accumulation of mutations in the
mitochondrial DNA seems to contribute to the process of aging as evident by
respiratory
chain function defects and mutations in mDNA with aging.
The methods and compounds of this invention can also be used to treat
neuromuscular disorders and epilepsy.
Creative Compounds Useful For Treating
Nervous System Diseases
Creative compounds useful in the present invention include compounds which
modulate one or more of the structural or functional components of the
creative
kinase/phosphocreatine system. Compounds which are effective for this purpose
include
creative, creative phosphate and analogs thereof, compounds which mimic their
activity,
and salts of these compounds as defined above. Exemplary creative compounds
are
described below.
Creative (also known as N-(aminoiminomethyl)-N-methylglycine;
methylglycosamine or N-methyl-guanido acetic acid} is a well-known substance.
(See,
The Merck Index, Eleventh Edition, No. 2570 ( 1989).
Creative is phosphorylated chemically or enzymatically by creative kinase to
generate creative phosphate, which also is well-known (see, The Merck Index,
No.
7315). Both creative and creative phosphate (phosphocreatine) can be extracted
from
animal tissue or synthesized chemically. Both are commercially available.
Cyclocreatine is an essentially planar cyclic analog of creative. Although
cyclocreatine is structurally similar to creative, the two compounds are
distinguishable
both kinetically and thermodynamically. Cyclocreatine is phosphorylated
efficiently by
creative kinase in the forward reaction both in vitro and in vivo. Rowley,
G.L., J. Am.

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Chem. Soc. 93: 5542-5551 (1971); McLaughlin, A.C. et. al., J. Biol. Chem. 247,
4382-4388 (1972).
The phosphorylated compound phosphocyclocreatine is structurally similar to
phosphocreatine; however, the phosphorous-nitrogen (P-N) bond of cyclocreatine
phosphate is more stable than that of phosphocreatine. LoPresti, P. and M.
Cohn,
Biochem. Biophys. Acta 998: 317-320 ( 1989); Annesley, T. M. and J. B. Walker,
J.
Biol. Chem. 253; 8120-8125, (1978); Annesley, T.M. and J.B. Walker. Biochem.
Biophys. Res. Commun. 74: 185-190 (1977).
Creatine analogs and other agents which act to interfere with the activity of
creatine biosynthetic enzymes or with the creatine transporter are useful in
the present
method of treating nervous system diseases. In the nervous system, there are
many
possible intracellular, as well as extracellular, sites for the action of
compounds that
inhibit, increase, or otherwise modify, energy generation through brain
creatine kinase
and/or other enzymes which are associated with it. Thus the effects of such
compounds
can be direct or indirect, operating by mechanisms including, but not limited
to,
influencing the uptake or biosynthesis of creatine, the function of the
creatine phosphate
shuttle, inhibiting the enzyme activity, or the activity of associated
enzymes, or altering
the levels of substrates or products of a reaction to alter the velocity of
the reaction.
Substances known or believed to modify energy production through the creatine
kinase/phosphocreatine system which can be used in the present method are
described
below. Exemplary compounds are shown in Tables 1 and 2.

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TABLE 1
CREATINE ANALOGS
NH NH O
O C~N~N H02C~N~N p~N ~ ~ -'NH
I H2 i ~ ~p
CH3 CH3 NH2
NH
N
~ ,- ~ ', +O
NH HO C~N~N p
HO C~N~NH 2 I ~ 0-PAN NH2
2 ~ CH2CH3 H CH
3
NH NH NH2
HO C~N~N
H02C~N~NH 2 I H2 H02C N ~ N
CHZCH2CH3
NH CH NH O CH3
HO C~ ~ HO C"N"N o0 ' NH2
2 N NH2 2 (R) I H2 ~p
CH3 NHZ
NH
NH rN NH 101 ~
H02C HO-P~N~NH
HOZC NH NH2 NH2 H U
NH
NH 101 ~
C HO-P~N~N
H02 (R)
NH2 H CH3

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TABLE 2
CREATINE ANALOGS
NH NH
HO C~N~N-PO~..~ H02C~N~N-POu
2 ( ~ 3' a2 I I 3"2
CH3 H CH3 H
NH
NH . ~H
HO C~N~N-POu
~ 2 3"2
HO C~N~N-POu
2 ~ 3"2 ~~CH3
NH
NH II H
H02C~N~N-PO H02C~N~N-P03H2
CH2CHZCH3
NH CH NH
H02C~ ~ HO C' _N' _N-PO
NH N-P03H2 2 (R) C H
H H3
NH ~N NH
HO2C (R)
H02C~NH N-P03H2 N-P03H2
H H
O NH H02C ~R~ N\ /NH
HO-P~N~NH POu N~-POu
3"2 ~ 3"2
H ~ H

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NH N
O ~ H2
.. ~ , O+
HO P N N-P03H2 O-P N ~NH2
H CH3 H H CH3
O NHz
p II~N~N-PO
O-P I I 3H2
H CH3 H
It will be possible to modify the substances described below to produce
analogs
which have enhanced characteristics, such as greater specificity for the
enzyme,
enhanced stability, enhanced uptake into cells, or better binding activity.
Compounds which modify the structure or function of the creative
kinase/creatine phosphate system directly or indirectly are useful in
preventing and/or
treating diseases of the nervous system characterized by up regulation or down
regulation of the enzyme system.
In diseases where the creative kinase/creatine phosphate system is down
regulated, for example, uncontrolled firing of neurons, molecules useful for
treating
these diseases include those that will up regulate the activity, or could
support energy
(ATP) production for a longer period of time. Examples include creative
phosphate and
related molecules that form stable phosphagens which support ATP production
over a
long period of time.
In diseases where the creative kinase/creatine phosphate system is up
regulated,
the molecules that are useful include those that will down regulate the
activity and/or
inhibit energy production (ATP).
Molecules that regulate the transporter of creative, or the association of
creative
kinase with other protein or lipid molecules in the membrane, the substrates
concentration creative and creative phosphate also are useful in preventing
and/or
treating diseases of the nervous system.
Compounds which are useful in the present invention can be inhibitors,
substrates or substrate analogs, of creative kinase, which when present, could
modify
energy generation or high energy phosphoryl transfer through the creative
kinase/phosphocreatine system. In addition, modulators of the enzymes that
work in
conjunction with creative kinase now can be designed and used, individually,
in

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combination or in addition to other drugs, to make control of the effect on
brain creatine
kinase tighter.
The pathways of biosynthesis and metabolism of creatine and creatine phosphate
can be targeted in selecting and designing compounds which modify energy
production
or high energy phosphoryl transfer through the creatine kinase system.
Compounds
targeted to specific steps may rely on structural analogies with either
creatine or its
precursors. Novel creatine analogs differing from creatine by substitution,
chain
extension, and/or cyclization may be designed. The substrates of
multisubstrate
enzymes may be covalently linked, or analogs which mimic portions of the
different
substrates may be designed. Non-hydrolyzable phosphorylated analogs can also
be
designed to mimic creatine phosphate without sustaining ATP production.
A number of creatine and creatine phosphate analogs have been previously
described in the literature or can be readily synthesized. Examples are these
shown in
Table I and Table 2. Some of them are slow substrates for creatine kinase.
Tables 1 and 2 illustrate the structures of creatine, cyclocreatine (1-
carboxymethyl-2-iminoimidazolidine), N-phosphorocreatine (N-phosphoryl
creatine),
cyclocreatine phosphate (3 -phosphoryl- 1 -carboxymethyl-2-iminoimidazolidine)
and
other compounds. In addition, 1-carboxymethyl-2-aminoimidazole, 1-
carboxymethyl-2
2-iminomethylimidazolidine, 1-carboxyethyl-2-iminoimidazolidine,
N-ethyl-N-amidinoglycine and b-guanidinopropionic acid are believed to be
effective.
Cyclocreatine (1-carboxymethyl-2-iminoimidazolidine) is an example of a class
of substrate analogs of creatine kinase, which can be phosphorylated by
creatine kinase
and which are believed to be active.
A class of creatine kinase targeted compounds are bi-substrate analogs
comprising an adenosine-like moiety linked via a modifiable bridge to a
creatine link
moiety (i.e., creatine or a creatine analog). Such compounds are expected to
bind with
greater affinity than the sum of the binding interaction of each individual
substrate (e.g.,
creatine and ATP). The modifiable bridge linking an adenosine-like moiety at
the
5'-carbon to a creatine like moiety can be a carbonyl group, alkyl (a branched
or straight
chain hydrocarbon group having one or more carbon atoms), or substituted alkyl
group
(an alkyl group bearing one or more functionalities, including but not limited
to
unsaturation, heteroatom-substituents, carboxylic and inorganic acid
derivatives, and
electrophilic moieties).
Another class of potential compounds for treating nervous system disorders is
designed to inhibit (reversibly or irreversibly) creatine kinase. The analogs
of creatine in
this class can bind irreversibly to the active site of the enzyme. Two such
affinity
reagents that have previously been shown to completely and irreversibly
inactivate
creatine kinase are epoxycreatine Marietta, M.A. and G:L. Kenyon J. Biol Chem.
254:

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1879-1886 (1979)) and isoepoxycreatine Nguyen, A.C.K., Ph.D. dissertation in
Pharmaceutical Chemistry, (University of California, San Francisco, 1983), pp.
112-205). There are several approaches to enhancing the specificity and hence,
the
eff cacy of active site-targeted irreversible inhibitors of creatine kinase,
incorporating an
electrophilic moiety. The effective concentration of a compound required for
inhibition
can be lowered by increasing favorable and decreasing unfavorable binding
contacts in
the creatine analog.
N-phosphorocreatine analogs also can be designed which bear nontransferable
moieties which mimic the N-phosphoryl group. These cannot sustain ATP
production.
Some currently preferred creatine compounds of this invention are those
encompassed by the general formula I:
Zt\
/C-X-A-Y
and pharmaceutically acceptable salts thereof, wherein:
a) Y is selected from the group consisting of -C02H-NHOH, -N02, -S03H,
-C(=O)NHS02J and -P(=O)(OH)(OJ), wherein J is selected from the group
consisting
of hydrogen, C1-C6 straight chain alkyl, C3-C6 branched alkyl, C2-C6 alkenyl,
C3-C6
branched alkenyl, and aryl;
b) A is selected from the group consisting of C, CH, Cl-CSalkyl, C2-CSalkenyl,
C2-CSalkynyl, and C1-CSalkoyl chain, each having 0-2 substituents which are
selected
independently from the group consisting of
1) K, where K is selected from the group consisting of C1-C6 straight alkyl,
C2-C6 straight alkenyl, C1-C6 straight alkoyl, C3-C6 branched alkyl, C3-C6
branched
alkenyl, and C4-C6 branched aIkoyl, K having 0-2 substituents independently
selected
from the group consisting of: bromo, chloro, epoxy and acetoxy;
2) an aryl group selected from the group consisting of a 1-2 ring carbocycle
and
a 1-2 ring heterocycle, wherein the aryl group contains 0-2 substituents
independently
selected from the group consisting of -CH2L and -COCH2L where L is
independently
selected from the group consisting of bromo, chloro, epoxy and acetoxy; and

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3) -NH-M, wherein M is selected from the group consisting of: hydrogen, C 1-C~
alkyl, C2-C4 alkenyl, C1-C4 alkoyl, C3-C4 branched alkyl, C3-C4 branched
alkenyl,
and C4 branched alkoyl;
c) X is selected from the group consisting of NR1, wherein R1 is selected from
the group
consisting of:
1 ) hydrogen;
2) K where K is selected from the group consisting of C 1 -C6 straight alkyl,
C2-C6 straight alkenyl, C1-Cg straight alkoyl, C3-C6 branched alkyl, C3-C6
branched
alkenyl, and C4-C6 branched alkoyl, K having 0-2 substituents independently
selected
from the group consisting of: bromo, chloro, epoxy and acetoxy;
3) an aryl group selected from the group consisting of a 1-2 ring carbocycle
and a
1-2 ring heterocycle, wherein the aryl group contains 0-2 substituents
independently
selected from the group consisting of: -CH2L and -COCH2L where L is
independently
selected from the group consisting of: bromo, chloro, epoxy and acetoxy;
4) a Cs-Cg a-amino-w-methyl-w-adenosylcarboxylic acid attached via the
w-methyl carbon;
5) 2 Cs-Cg a-amino-w-aza-w-methyl-w-adenosylcarboxylic acid attached via the
w-methyl carbon; and
6) a Cs-Cg a-amino-w-thia-w-methyl-w-adenosylcarboxylic acid attached via the
w-methyl carbon;
d) Zl and Z2 are chosen independently from the group consisting of: =O, -NHR2,
-CH2R2, -NR20H; wherein Z1 and Z2 may not both be =0 and wherein R2 is
selected
from the group consisting of:
1)hydrogen;

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2) K, where K is selected from the group consisting of: C1-CS straight alkyl;
C2-C6 straight alkenyl, C1-C6 straight alkoyl, C3-C6 branched alkyl, C3-C6
branched
alkenyl, and C4-C6 branched alkoyl, K having 0-2 substituents independently
selected
from the group consisting of bromo, chloro, epoxy and acetoxy;
3) an aryl group selected from the group consisting of a 1-2 ring carbocycle
and a
1-2 ring heterocycle, wherein the aryl group contains 0-2 substituents
independently
selected from the group consisting of: -CH2L and -COCH2L where L is
independently
selected from the group consisting of bromo, chloro, epoxy and acetoxy;
4) 2 C4-Cg a-amino-carboxylic acid attached via the w-carbon;
5) B, wherein B is selected from the group consisting of -C02H-NHOH,
-S03H, -N02, OP(=O)(OH)(OJ) and -P(=O)(OH)(OJ), wherein J is selected from the
group consisting of hydrogen, C1-C6 straight alkyl, C3-C6 branched alkyl, C2-
C6
alkenyl, C3-C6 branched alkenyl, and aryl, wherein B is optionally connected
to the
nitrogen via a linker selected from the group consisting of C1-C2 alkyl, C2
alkenyl, and
C1-C2 alkoyl;
6) -D-E, wherein D is selected from the group consisting of C1-C3 straight
alkyl, C3 branched alkyl, C2-C3 straight alkenyl, C3 branched alkenyl, C 1 -C3
straight
alkoyl, aryl and aroyl; and E is selected from the group consisting of: -
(P03)nNMP,
where n is 0-2 and NMP is ribonucleotide monophosphate connected via the
5'-phosphate, 3'-phosphate or the aromatic ring of the base; -
[P(=O)(OCH3)(0)]m-Q,
where m is 0-3 and Q is a ribonucleoside connected via the ribose or the
aromatic ring of
the base; -[P(=O)(OH)(CH2)]m-Q, where m is 0-3 and Q is a ribonucleoside
connected
via the ribose or the aromatic ring of the base; and an aryl group containing
0-3
substituents chosen independently from the group consisting of: Cl, Br, epoxy,
acetoxy,
-OG, -C(=O)G, and -C02G, where G is independently selected from the group
consisting of: C1 -C6 straight alkyl, C2-C6 straight alkenyl, C1-C6 straight
alkoyl,
C3-C6 branched alkyl, C3-C6 branched alkenyl, C4-Cg branched alkoyl, wherein E
may
be attached to any point to D, and if D is alkyl or alkenyl, D may be
connected at either
or both ends by an amide linkage; and

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7) -E, wherein E is selected from the group consisting of -(P03)nNMP,
where n is 0-2 and NMP is a ribonucleotide monophosphate connected via the
5'-phosphate, 3'-phosphate or the aromatic ring of the base; -
[P(=O)(OCH3)(0)]m-Q,
where m is 0-3 and Q is a ribonucleoside connected via the ribose or the
aromatic ring of
the base; -[P(=O)(OH)(CH2)]m-Q, where m is 0-3 and Q is a ribonucleoside
connected
via the ribose or the aromatic ring of the base; and an aryl group containing
0-3
substituents chose independently from the group consisting of: Cl, Br, epoxy,
acetoxy,
-OG, -C(=O)G, and -C02G, where G is independently selected from the group
consisting of: Cl-C6 straight alkyl, C2-C6 straight alkenyl, Cl-C6 straight
alkoyl,
C3-C6 branched alkyl, C3-C6 branched alkenyl, C4-C6 branched alkoyl; and if E
is aryl,
E may be connected by an amide linkage;
e) if Rl and at least one R2 group are present, R1 may be connected by a
single or
double bond to an R2 group to form a cycle of 5 to 7 members;
fJ if two R2 groups are present, they may be connected by a single or a double
bond
to form a cycle of 4 to 7 members; and
g) if Rl is present and Zl or Z2 is selected from the group consisting of -
NHR2, -CH2R2
and -NR20H, then Rl may be connected by a single or double bond to the carbon
or
nitrogen of either Zl or Z2 to form a cycle of 4 to 7 members.
Creatine, creatine phosphate and many creatine analogs, and competitive
inhibitors are commercially available. Additionally, analogs of creatine may
be
synthesized using conventional techniques. For example, creatine can be used
as the
starting material for synthesizing at least some of the analogs encompassed by
formula I.
Appropriate synthesis reagents, e.g. alkylating, alkenylating or alkynylating
agents may
be used to attach the respective groups to target sites. Alternatively,
reagents capable of
inserting spacer groups may be used to alter the creatine structure. Sites
other than the
target site are protected using conventional protecting groups while the
desired sites are
being targeted by synthetic reagents.
If the creatine analog contains a ring structure, then the analog may be
synthesized in a manner analogous to that described for cyclocreatine (Wang,
T., J. Org.
Chem. 39:3591-3594 (1974)). The various other substituent groups may be
introduced
before or after the ring is formed.

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Many creative analogs have been previously synthesized and described (Rowley
et al., J. Am. Chem. Soc. 93:5542-5551 (1971); McLaughlin et al., J. Biol.
Chem.
247:4382-4388 (1972) Nguyen, A.C.K., "Synthesis and enzyme studies using
creative
analogs", Thesis, Dept. of Pharmaceutical Chemistry, Univ. Calif., San
Francisco
(1983); Lowe et al., J. Biol. Chem. 225:3944-3951 (1980); Roberts et al, J.
Biol. Chem.
260:13502-13508 (1985); Roberts et al., Arch. Biochem. Biophys. 220:563-571
(1983),
and Griffiths et al, J. Biol. Chem. 251 :2049-2054 (1976)). The contents of
all of the
forementioned references are expressly incorporated by reference. Further to
the
forementioned references, Kaddurah-Daouk et al. (W092/08456; W090/09192; U.S.
5,324,731; U.S. 5,321,030) also provide citations for the synthesis of a
plurality of
creative analogs. The contents of all the aforementioned references and
patents are
incorporated herein by reference.
Creative compounds which currently are available or have been synthesized
include, for example, creative, b-guanidinopropionic acid, guanidinoacetic
acid, creative
phosphate disodium salt, cyclocreatine, homocyclocreatine, phosphinic
creative,
homocreatine, ethylcreatine, cyclocreatine phosphate dilithium salt and
guanidinoacetic
acid phosphate disodium salt, among others.
Creative phosphate compounds also can be synthesized chemically or
enzymatically. The chemical synthesis is well known. Annesley, T.M. Walker,
J.B.,
Biochem. Biophys. Res. Commun., (1977), 74, 185-190; Cramer, F., Scheiffele,
E.,
Vollmar, A., Chem. Ber., (1962), 95, 1670-1682.
Salts of the products may be exchanged to other salts using standard
protocols.
The enzymatic synthesis utilizes the creative kinase enzyme, which is
commercially
available, to phosphorylate the creative compounds. ATP is required by
creative kinase
for phosphorylation, hence it needs to be continuously replenished to drive
the reaction
forward. It is necessary to couple the creative kinase reaction to another
reaction that
generates ATP to drive it forward. The purity of the resulting compounds can
be
confirmed using known analytical techniques including 1H NMR, 13CNMR Spectra,
Thin layer chromatography, HPLC and elemental analysis.
Existing Therapeutic Agents for
Neurodegenerative Diseases
Therapeutic agents for treatment of neurodegenerative disease which are useful
in combination with creative compounds or creative compounds and
neuroprotective
agents are described below.
Suitable therapeutic drugs for neurodegenerative diseases include those which
have been approved by, for example, the United States Food and Drug
Administration.

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Representative drugs useful in treatment of Alzheimer's disease include Cognex
(tacrine)
manufactured by Parke Davis which is a first generation acetylcholinesterase
inhibitor
and Aricept (donepizil) manufactured by Eisai which is a second generation
acetylcholinesterase inhibitor.
Suitable drugs for treatment of Parkinson's Disease include Sinemet
(carbidopa/levidopa) and Sinemet CR (carbidopa/levidopa sustained release)
manufactured by DuPont Pharma. Levodopa is a metabolic precursor of dopamine
that
crosses the blood-brain barrier. Carbidopa inhibits conversion of levodopa
before it
crosses the blood-brain barrier. Permax (pergolide mesylate), manufactured by
Athena,
and Parlodel (bromocriptine mesylate), manufactured by Novartis, are
therapeutic agents
for treatment of Parkinson's Disease and are dopamine receptor agonists, often
used as
an adjunct to Sinemet. Eldepryl (selegiline}, manufactured by Somerset, is yet
another
therapeutic agent for treatment of Parkinson's Disease and inhibits monoamine
oxidase
and is used as an adjunctive therapy. Symmetrel (amantadine), manufactured by
DuPont
Pharma, has an unknown mechanism of treatment for Parkinson's Disease. Artane
(trihexyphenidyl hydrochloride}, manufactured by Lederle, also a suitable
therapeutic
agent is a muscarinic antagonist and is used as an adjunctive therapy.
An example of a therapeutic drug for treatment of ALS is Rilutek (riluzole),
manufactured by Rhone-Poulenc Rorer. Rilutek elicits an inhibitory effect on
glutanate
release and has various neuroprotective effects, however, the mode of its
action is
unknown.
Neuroprotective Agents Useful For Treating
Nervous System Diseases
Neuroprotective agents include those compositions which provide
neuroprotection, e.g., approved drugs for the treatment or prevention of
neurodegenerative diseases such as Riluzole, Cognex, Aricept, Sinmet, Sinmet
CR,
Permax, Parlodel, Elepryl, Symmetrel, Artane); glutamate excitotoxicity
inhibitors
(such as glutamate uptake and biosynthesis modulation with compounds like
gabapentin
and Riluzole); growth factors like CNTF, BDNF, IGF-1; nitric oxide synthase
inhibitors; cyclo-oxygenase inhibitors such as aspirin; ICE inhibitors;
Neuroimmunophilins; N-acetylcysteine and procysteine; antioxidants, energy
enhancers,
vitamins and cofactors (such as spin traps, CoQlO, carnitine, nicotinamide,
Vit E or D)
and lipoic acid.
ATP Enhancing Agents Useful for Electron Transport
ATP enhancing agents include those compounds which facilitate ATP
production. These agents can be critical in the function of electron transport
and

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oxidative phosphorylation and hence ATP production and neuronal cell survival.
Examples include:
Nicotinamide/Riboflavin:
Riboflavin and nicotinamide are water soluble vitamins and components of
coenzymes critical in the function of electron transport and oxidative
phosphorylation
and hence ATP production. The water soluble vitamins are referred to as the
vitamin B
complex. Riboflavin (vitamin B2) is a precursor of FAD, and niacin is the
precursor of
Nicotinamide adenine dinucleotide. Nicotinamide adenine dinucleotide is a
major
electron acceptor in the oxidation of fuel molecules. The reactive part of
NAD+ is the
nicotinamide ring. In the oxidation of substrates the nicotinamide ring of
NAD+ accepts
a hydrogen ion and two electrons which are equivalent to a hydride ion. The
reduced
form of this carrier is called NADH. The other major electron carrier in the
oxidation of
fuel molecules is flavin adenine dinucleotide. FAD like NAD+ is a two electron
acceptor. Hence the molecules riboflavin and nicotinamide are used as
supplements to
drive effectively oxidative phosphorylation and could have significant
protective effects
in stress conditions or disease states where energy production and oxidative
phosphorylation are compromised.
Nicotinamide is a B vitamin and is a major component ofNAD, and NADP
which are critical components in the regulation of electron transport chain
and energy
production in the mitochondria. Nicotinamide is the amide of nicotinic acid,
is a
crystalline compound of the vitamin B complex, is convertible into nicotine
acid in the
body. Nicotinic acid is a group of vitamins of the B complex, central for
growth and
health in many animals and important in protein and carbohydrate metabolism.
It is
found in meat, liver, wheat germ, milk eggs. Also, Niacin is converted to
nicotinamide
in the body.
Treatment with nicotinamide in combination with riboflavin (Penn et.al.,
Neurology, 42: 2147-2152, 1992; Bernsen et.al., J. Neurol Sci. 118: 181-187,
1993)
result in both biochemical and clinical improvement for patients with
mitochondria)
disorders. The combination of nicotinamide and coenzyme Q10 were shown to
attenuate
malonate induced energy defects and attenuate the striatial lesions produced
by this
compound, i.e., an animal model of Huntigton's disease (Beat et.al., Annals of
Neurology, 26: 882-888, 1994). Amounts used were Q10 100-300 mg/kg/day,
nicotinamide 500 mg/kg/day, and riboflavin 15 mg/kg/day.

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Co-Enzyme Qs (CoQs):
A Cogs is a member of the family of co-enzyme Qs wherein the "s" is the
number of isoprenoid units attached to the quinone ring. CoQlO is a preferred
Cogs of
the present invention. CoQlO is present in virtually all living cells.
Although a
molecular structure varies among different types of organisms, the chemical
structure of
CoQlO (2,3 dimethoxy-5 methyl-6-decaprenyl benzoquinone) consists of a quinone
ring
(a molecular structure of carbon, hydrogen, and oxygen) with a long side
chain. The
body of the molecule is always the same but the number of the isoprene units
(a 5 carbon
chemical unit) attached to the quinone ring varies (human CoQ 10 has 10 iso-
prenoid
units) the side chain is highly fat soluble which allows coql0 to lodge firmly
in
membranes inside cells. CoQlp is a large lipophilic fat soluble nutrient with
a mol wt.
of 862D. It is very soluble in chloroform and carbon tetrachloride and
insoluble in
water. CoQIO is poorly absorbed unless it is specially prepared by
solubilizing-
emmulsifing in suitable oils or emmulsified in a silica base excipient
containing a non-
I 5 ionic surfactant. Multi approaches have been developed to enhance the bio-
availability
of the compound such as the use of oily preparations to bypass the liver.
CoQlO is an essential nutrient that is a co-factor in the mitochondria)
electron
transport chain, the biochemical pathway in cellular respiration in which ATP
and
metabolic energy is derived, since all cellular functions depend on energy CoQ
I O seems
to be essential for the health of human tissue. Additionally, CoQlO similar to
Vitamin
E, and K has anti-oxidant activity and scavenges free radical which could add
to it's
benefit to minimize injury for example to neuronal cells. Diets could be
deficient in
providing sufficient amounts of CoQ 10 suggesting that supplementation with
this
compound could be of benefit in preserving tissue.
CoQlO was first isolated from beef heart mitochondria by Dr. Frederick Crane
in
1957 (Crane et al., Biochimica et Biophys. Acta, Vo125:220-221, 1957). In 1958
Prof.
Karl Folkers and co-workers at Merck, Inc. determined the precise chemical
structure of
CoQlp: 2,3 dimethoxy-S methyl-6-decaprenyl benzoquinone, synthesized it and
were
the first to produce it by fermentation. In the mid 1960's Prof. Yamamura of
Japan was
the first to use CoQ7 a related compound to treat a human disease (congestive
heart
failure). Multi clinical trials with CoQlO followed.
Improved cardiovascular morbidity and mortality have been observed in several
clinical studies using CoQlO as a supplement (Serebruany et al., J.
Cardiovascular
Pharmacology 28(2):1775-181, 1996). Pretreatment with CoQIO at 150 mg/day for
7
days suggested some protective benefit for patients undergoing routine
vascular

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procedures requiring abdominal aortic cross clamping by attenuating the degree
of
peroxidative damage (Chello et al., J. of Cardiovascular Surgery 37(3):229-
235, 1996).
Benefit to patients with cardiomyopathy has been suggested with the use of CoQ
10 at
100 mg/day for several weeks to years (Manzoli et al, It. J. Tiss. Reac.
12(3):173-178,
1990; Langsjoen. et al., Int. J. Tiss. Reac. 12(3):163-168, 1990; Langsjoen.
et al., Am. J.
Cardiol. (65):521-523, 1990, Langsjoen. et al., nt. J. Tiss. Reac. 12(3):169-
171, 1990;
Morisco et al., Clin Invest. 71:S 134-5136, 1993).
Patients with mitochondria) myopathies placed on CoQ 1 p supplementation at
100-150 mg/day, for extended periods of time, showed benefit in reversing
abnormal
biochemical profiles and muscle function (Nakamura et al., Electromyography
and
Clinical Neurophysiology 35(6):365-370, 1995, Gold et al., Eur. Neurology
36(4):191-
196, 1996, Ikerjiri et al. Neurol. 47(2):583-585, 1996). Also patients with
mitochondria)
myopathies secondary to HIV infection and treatment with AZT might beneft from
CoQlO supplementation (Dalakas et al., N Eng J Med. 322:1098-1105, 1990).
Improved
physical performance in patients with muscle dystrophies was noted upon
supplementation with CoQlO (Folkers et al., Biochimica et Biophysica Acta-
Molecular
Basis of Disease 1271(1):281-286, 1995). The combination of CoQlp and
Nicotinamide
blocked striatal lesions produced by the mitochondria) toxin Malonate, an
animal model
of Huntington's Disease (Beat et al., Ann. Neuro 36(6):882-888, 1994). The
combination of CoQ 10 and Nicotinamide and free radical spin traps protected
against
MPTP neurotoxicity, an animal model of Parkinson's Disease (Schulz et al.,
Exp.
Neurol. 132:279-283, 1995).
Free Radical Spin Traps:
Free radicals are formed as food and oxygen are metabolized to produce energy.
These radicals can oxidize and kill cells. Oxidation is a chemical reaction in
which a
molecule transfers one or more electrons to another. Stable molecules usually
have
matched pairs of protons and electrons. In certain reactions, a free radical
can be formed
having unpaired electrons. Free radicals tend to be highly reactive, oxidizing
agents.
Free radicals can kill cells by damaging cell membranes, cytoskeleton and
sensitive
nuclear and mitochondria) DNA. Such intracellular damage can lead to the
increase in
calcium, increase in damaging proteases and nucleases and production of
interferons,
TNF-a and other tissue damaging mediators which lead to disease if
overexpressed in
response to oxidative stress. When free radicals interact with non-radicals,
the result is
usually a chain reaction. Only when two radicals meet or when antioxidants
quench the
reaction is the cascade of damage terminated. The most common reactive oxygen

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species {ROS) produced in vivo are hydrogen peroxide H202, hydroxyl OH,
superoxide
02, perhydroxyl H02, nitrogen oxide NO, and alkoxyl RO, and peroxyl ROO
radicals.
In normal healthy individuals this process is offset by endogenous
antioxidants
and cellular repair mechanisms. However as we age and in certain diseases, the
process
can fall out of balance resulting in delitating and potentially fatal
consequences.
Oxidation is important factor in many diseases and disorders such as
Parkinson's disease
and Alzheimer's disease, ischemia reperfusion injury associated with stroke
and heart
attack, and inflammatory conditions such as arthritis and ocular inflammation,
AIDS
dementia complex, inflammatory bowel disease and rational neovascularization,
and
multiple sclerosis.
Oxygen breathing animals have developed powerful antioxidant defense systems
and cellular repair mechanisms to control this damage. Enzymes such as
superoxide
dismutase, catalse and glutathione peroxidase and vitamins such as tocopherol,
ascorbate
and carotene act to quench radical chain reactions. In general many of these
natural
molecules alone do not have great activity when given as supplements because
they have
to be produced within the cells to be effective in disease prevention.
Spin traps are chemical compounds that can protect cells from damaging effects
of free radicals and hence slow or reverse the oxidation damage associated
with these
conditions. Suitable spin traps include PBN, S-PBN, DMPO, TEMPOL, azulenyl
based
spin traps, MDL, etc.
In an animal model of Parkinson's disease, nicotinamide or the free radical
spin
trap N-tert-a-(2-sulfophenyl) nitrone were effective in inhibiting moderate
dopamine
depletion (Schulz et al., Experimental Neurology 132, 279-283, 1995). In the
same
study, Q10 and nicotinamide protected against both mild and moderate depletion
of
dopamine. These results show that agents which improve mitochondria) energy
production like Q 10 and nicotinamide and the free radical scavengers can
attenuate mild
to moderate MPTP neurotoxicity.
Several free radical spin trap compounds can exert neuroprotective effects
against both excitotoxicity and mitochondria) toxins in vivo.
I ,_C'arnitine:
Carnitine is an important cofactor for normal cellular metabolism. Optimal
utilization of fuel substrates for ATP generation is dependent on adequate
carnitine
stores. Fatty acids are activated on the outer mitochondria) membrane, whereas
they are
oxidized in the mitochondria) matrix. Long chain acyl CoA molecules do not
readily
traverse the inner mitochondria) membrane, and so a special transport
mechanism is
needed. Activated long chain fatty acids are carried across the inner
mitochondria)

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membrane by carnitine. The acyl group is transferred from the sulfur atom of
CoA to
the hydroxyl group of carnitine to form acyl carnitine, which diffuses across
the inner
mitochondria) membrane. On the matrex side of this membrane the acyl group is
transferred back to CoA; which is thermodynamically feasible because of the O-
acyl link
in carnitine has high transfer potential. Oxidation of long chain fatty acids
provides an
excellent source of energy. Deficiencies of carnitine might result in impaired
flow of
metabolities form one compartment of a cell to another which can result in
disease.
The supplementation of L Carnitine was shown to have some benefit to chronic
hemodialysis patients. patients with cardiovascular diseases, muscle diseases,
chronic
fatigue, diabetic neuropathies, AIDS patients. Typical doses are 20-30 mg/Kg.
Anti-oxidants:
Anti-oxidants include those species of compounds which inhibit or prevent
oxidation of tissues, such as vitamin E, alpha-omega fatty acids, BHP, etc.
such as those
known in the art.
Reactive oxygen species are thought to be involved in a number of types of
acute
and chronic pathologic conditions in the brain and neural tissue. The
metabolic
antioxidant alpha-lipoate (thioctic acid, 1, 2-dithiolane-3-pentanoic acid; 1,
2-dithiolane-
3 valeric acid; and 6, 8-dithiooctanoic acid) is a low molecular weight
substance that is
absorbed from the diet and crosses the blood-brain barrier. Alpha-lipoate is
taken up and
reduced in cells and tissues to dihydrolipoate, which is also exported to the
extracellular
medium; hence, protection is afforded to both intracellular and extracellular
environments. Both alpha-lipoate and especially dihydrolipoate have been shown
to be
potent antioxidants, to regenerate through redox cycling other antioxidants
like vitamin
C and vitamin E, and to raise intracellular glutahione levels. Thus, it
appears an ideal
substance in the treatment of oxidative brain and neural disorders involving
free-radical
processes. Examination of current research reveals protective effects of these
compounds in cerebral ischemia-reperfusion, excitotoxic amino acid brain
injury,
mitochondria) dysfunction, diabetes and diabetic neuropathy, inborn errors of
metabolism, and other causes of acute or chronic damage to brain or neural
tissue. Very
few neuropharinacological intervention strategies are currently available for
the
treatment of stroke and numerous other brain disorders involving free radical
injury. It
is believed that the various metabolic antioxidant properties of alpha-lipoate
relate to its
possible therapeutic roles in a variety of brain and neuronal tissue
pathologies: thiols are
central to antioxidant defense in brain and other tissues. The most important
thiol
antioxidant, glutahione, cannot be directly administered, whereas alpha-lipoic
acid can.
In vitro, animal, and preliminary human studies indicate that alpha-lipoate
may be
effective in numerous neurodegenerative disorders.

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Utility
In the present invention, the combinations of creative compounds and
neuroprotective agents can be administered to an individual (e.g., a mammal),
alone or in
combination with another compound, for the treatment of diseases of the
nervous
system. As agents for the treatment of diseases of the nervous system,
creative
compounds can interfere with creative kinase/phosphocreatine functions,
thereby
preventing, ameliorating, arresting or eliminating direct and/or indirect
effects of disease
which contribute to symptoms such as paraplegia or memory impairment. Other
compounds which can be administered together with the creative compounds
include
neurotransmitters, neurotransmitter agonists or antagonists, steroids,
corticosteroids
(such as prednisone or methyl prednisone) immunomodulating agents (such as
beta-
interferon), immunosuppressive agents (such as cyclophosphamide or
azathioprine),
nucleotide analogs, endogenous opioids, or other currently clinically used
drugs. When
co-administered with creative compounds, these agents can augment interference
with
creative kinase/phosphocreatine cellular functions, thereby preventing,
reducing, or
eliminating direct and/or indirect effects of disease.
A variety of diseases of the nervous system can be treated with creative or
creative analogs in combination with neuroprotective agents, including but not
limited to
those diseases of the nervous system described in detail above. Others include
bacterial
or fungal infections of the nervous system. These creative or analog
combinations can
be used to reduce the severity of a disease, reduce symptoms of primary
disease
episodes, or prevent or reduce the severity of recurrent active episodes.
Creative,
creative phosphate or analogs such as cyclocreatine and cyclocreatine
phosphate can be
used to treat progressive diseases. Many creative analogs can cross the blood-
brain
barrier. For example, treatment can result in the reduction of tremors in
Parkinson's
disease, and other clinical symptoms.
Modes of Administration
The creative compound and neuroprotective agent can be administered to the
afflicted individual alone or in combination with another creative analog or
other agent.
The combinations can be administered as pharmaceutically acceptable salts in a
pharmaceutically acceptable carrier, for example. The combinations may be
administered to the subject by a variety of routes, including, but not
necessarily limited
to, oral (dietary), transdermal, or parenteral (e.g., subcutaneous,
intramuscular,
intravenous injection, bolus or continuous infusion) routes of administration,
for
example. An effective amount (i.e., one that is sufficient to produce the
desired effect in
an individual) of a composition comprising a creative analog and a
neuroprotective agent

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is administered to the individual. The actual amount of drug to be
administered will
depend on factors such as the size and age of the individual, in addition to
the severity of
symptoms, other medical conditions and the desired aim of treatment.
Previous studies have described the administration and efficacy of creatine
compounds in vivo. For example, creatine phosphate has been administered to
patients
with cardiac diseases by intravenous injection. Up to 8 grams/day were
administered
with no adverse side effects. The efficacy of selected creatine kinase
substrate analogs
to sustain ATP levels or delay rigor during ischemic episodes in muscle has
been
investigated. On one study, cyclocreatine was fed to mice, rats and chicks,
and appeared
to be well-tolerated in these animals. Newly hatched chicks were fed a diet
containing
1 % cyclocreatine. In the presence of antibiotics, the chicks tolerated 1 %
cyclocreatine
without significant mortality, although the chicks grew more slowly than
control chicks
(Griffiths, G. R. and J. B. Walker, J. Biol. Chem. 251 (7): 2049-2054 (1976)).
In
another study, mice were fed a diet containing 1 % cyclocreatine for 10 days
(Annesley,
T. M. and J. B. Walker, J. Biol. Chem. 253(22): 8120-8125 (1978)).
Cyclocreatine has
been feed to mice at up to 1 % of their diet for 2 weeks or for over 4 weeks
without gross
adverse effects. Lillie et al., Cancer Res., 53: 3172-3178(1993). Feeding
animals
cyclocreatine (e.g., 1% dietary) has been shown to lead to accumulation of
cyclocreatine
in different organs in mM concentrations. For example, cyclocreatine was
reported to be
taken up by muscle, heart and brain in rats receiving dietary 1 %
cyclocreatine. Griffiths,
G. R. and J. B. Walker, J. Biol. Chem. 251 (7): 2049-2054 ( 1976). As shown
previously,
antiviral activity of cyclocreatine is observed on administering 1 % dietary
cyclocreatine.
Many of the above-referenced studies show that creatine analogs are been shown
to be
capable of crossing the blood-brain barrier.
The creatine compound and neuroprotective agent combination can be
formulated according to the selected route of administration (e.g., powder,
tablet,
capsule, transdermal patch, implantable capsule, solution, emulsion). An
appropriate
composition comprising a creatine analog and neuroprotective agent can be
prepared in a
physiologically acceptable vehicle or carrier. For example, a composition in
tablet form
can include one or more additives such as a filler (e.g., lactose), a binder
(e.g., gelatin,
carboxymethylcellulose, gum arabic), a flavoring agent, a coloring agent, or
coating
material as desired. For solutions or emulsions in general, carriers may
include aqueous
or alcoholic/aqueous solutions, emulsions or suspensions, including saline and
buffered
media. Parenteral vehicles can include sodium chloride, solution, Ringer's
dextrose,
dextrose and sodium chloride, lactated Ringer's or fixed oils. In addition,
intravenous
vehicles can include fluid and nutrient replenishers, and electrolyte
replenishers, such as
those based on Ringer's dextrose. Preservatives and other additives can also
be present.

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For example, antimicrobial, antioxidant, chelating agents, and inert gases can
be added.
(See, generally, Remington's Pharmaceutical Sciences, 16th Edition, Mack, Ed.,
1980).
The term "administration" is intended to include routes of administration
which
allow the creative compound/neuroprotective agent to perform their intended
functions)
of preventing, ameliorating, arresting, and/or eliminating diseases) of the
nervous
system in a subject. Examples of routes of administration which may be used
include
injection (subcutaneous, intravenous, parenterally, intraperitoneally, etc.),
oral,
inhalation, transdermal, and rectal. Depending on the route of administration,
the
creatine/neuroprotective agent may be coated with or in a material to protect
it from the
natural conditions which may detrimentally effect its ability to perform its
intended
function. The administration of the creatine/neuroprotective agent is done at
dosages
and for periods of time effective to reduce, ameliorate or eliminate the
symptoms of the
nervous system disorder. Dosage regimes may be adjusted for purposes of
improving
the therapeutic or prophylactic response of the compound. For example, several
divided
doses may be administered daily or the dose may be proportionally reduced as
indicated
by the exigencies of the therapeutic situation.
In addition, the methods of the instant invention comprise creative compounds
effective in crossing the blood-brain barrier.
The creative compounds/neuroprotective agents of this invention may be
administered alone or as a mixture with other creative compounds, or together
with an
adjuvant or other drug. For example, the creative compound/neuroprotective
agent may
be coadministered with other different art-recognized moieties such as
nucleotides,
neurotransmitters, agonists or antagonists, steroids, immunomodulators,
immunosuppressants, vitamins, endorphins or other drugs which act upon the
nervous
system or brain.
Creative Kinase Isoenzymes in the Brain
Cells require energy to survive and to carry out the multitude of tasks that
characterize biological activity. Cellular energy demand and supply are
generally
balanced and tightly regulated for economy and efficiency of energy use.
Creative
kinase plays a key role in the energy metabolism of cells with intermittently
high and
fluctuating energy requirements such as skeletal and cardiac muscle, brain and
neural
tissues, including, for example, the retina, spermatozoa and electrocytes. As
stated
above, the enzyme catalyzes the reversible transfer of the phosphoryl group
from
creative phosphate to ADP, to generate ATP. There are mufti-isoforms of
creative
kinase (CK) which include muscle (CK-MM), brain (CK-BB) and mitochondria)
(CK-Mia, CK-Mib} isoforms.

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Experimental data suggest that CK is located near the sites in cells where
energy
generation occurs; e.g., where force generation by motor proteins takes place,
next to ion
pumps and transporters in membranes and where other ATP-dependent processes
take
place. It seems to play a complex mufti-faceted role in cellular energy
homeostasis. The
creative kinase system is involved in energy buffering/energy transport
activities. It also
is involved in regulating ADP and ATP levels intracellularly as well as
ADP/ATP ratios.
Proton buffering and production of inorganic phosphate are important parts of
the
system.
In the brain, this creative kinase system is quite active. Regional variations
in
CK activity with comparably high levels in cerebellum were reported in studies
using
native isoenzyme electrophoresis, or enzymatic CK activity measurements in
either
tissue extracts or cultured brain cells. Chandler et al. Stroke, 19: 251 -255
(1988),
Maker et al. Exp. Neurol., 38: 295-300 (1973), Manos et al. J. Neurol. Chem.,
_56:
21 O 1-2I 07 ( 1991 ). In particular, the molecular layer of the cerebellar
cortex contains
high levels of CK activity (Maker et a1. id. (1973) Kahn Histochem., 48: 29-32
(1976)
consistent with the recent 3'P-NMR findings which indicate that gray matter
shows a
- higher flux through the CK reaction and higher creative phosphate
concentrations as
compared to white matter (Cadoux-Hudson et al. FASEBJ., 3: 2660-2666 (1989),
but
also high levels of CK activity were shown in cultured oligodendrocytes (Manos
et al.
id. (1991), Molloy et al. J. Neurochem., 59: 1925-1932 (1992), typical glial
cells of the
white matter. The brain CK isoenzyme CK-BB is the major isoform found in the
brain.
Lower amounts of muscle creative kinase (CK-MM) and mitochondria) creative
kinase
(CK-Mi) are found.
Localization and Function of CK Isoenzymes
in Different Cells of the Nervous System
Brain CK (CK-BB) is found in all layers of the cerebellar cortex as well as in
deeper nuclei of the cerebellum. It is most abundant in Bergmann glial cells
(BGC) and
astroglial cells, but is also found in basket cells and neurons in the deeper
nuclei.
Hemmer et al., Eur. J. Neuroscience, 6: 538-549 (1994), Hemmer et al. Dev.
Neuroscience, 15: 3-5 (1993). The BGC is a specialized type of astroglial
cell. It
provides the migratory pathway for granule cell migration from the external to
the
internal granule cell layer during cerebellar development. Another main
function of
these cells is the proposed ATP-dependent spatial buffering of potassium ions
released
during the electrical activity of neurons (Newman et al. Trends Neuroscience,
_8: 156-159
( 1985), Reichenbach, Acad. Sci New York, ( I991 ), pp. 272-286. Hence, CK-BB
seems
to be providing energy (ATP) for migration as well as K+ buffering through
regulation

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of the Na+/K+ ATPase. The presence of CK-BB in astrocytes (Manos et al. id.
1991,
Hemmer et al. id. 1994, Hemmer et al. id. 1993) may be related to the energy
requirements of these cells for metabolic interactions with neurons; e.g.,
tricarboxylic
acid cycle (TCA) metabolite and neurotransmitter trafficking. Hertz, Can J.
Physiol.
Pharmacol., 70: 5145-S 157 ( 1991 ).
The Purkinje neurons of the cerebellum play a very important role in brain
function. They receive excitatory input from parallel fibers and climbing
fibers, they
represent the sole neuronal output structures of the cerebellar cortex.
Calcium mediated
depolarizations in Purkinje cell dendrites are thought to play a central role
in the
mechanism of cerebellar motoric learning. Ito Con. Opin. Neurobiol., 1: 616-
620
(1991). High levels of muscle CK (CK-MM) were found in Purkinje neurons.
Hemmer
et al. id. (1994), Hemmer et al., id. (1993). There is strong evidence to
support that
CK-MM is directly or indirectly coupled to energetic processes needed for Cap
homeostasis or to cellular processes triggered by this second messenger.
The glomerular structures of the cerebellum contain high levels of CK-BB and
mitochondrial CK (CK-Mi). Large amounts of energy are needed in these
structures for
restoration of potassium ion gradients partially broken down during neuronal
excitation
as well as for metabolic and neurotransmitter trafficking between glial cells
and neurons.
Hertz et al., id. ( 1991 ). The presence of CK in these structures may be an
indication that
part of the energy consumed in these giant complexes might be supported by the
creative
kinase system.
In neurons, CK-BB is found in association with synaptic vesicles (Friedhoff
and
Lerner, Life Sci., 20: 867-872 (1977) as well as with plasma membranes (Lim et
al., J.
Neurochem., 41: 1177-1182 (1983)).
There is evidence to suggest that CK is bound to synaptic vesicles and to the
plasma membrane in neurons may be involved in neurotransmitter release as well
as in
the maintenance of membrane potentials and the restoration of ion gradients
before and
after stimulation. This is consistent with the fact that high energy turnover
and
concomitantly high CK concentrations have been found in those regions of the
brain that
are rich in synaptic connections; e.g., in the molecular layer of the
cerebellum, in the
glomerular structures of the granule layer and also in the hippocampus. The
observation
that a rise in CK levels observed in a fraction of brain containing nerve
endings and
synapses, parallels the neonatal increase in Na+/K+ ATPase is also suggestive
that
higher levels of creative phosphates and CK are characteristic of regions in
which energy
expenditure for processes such as ion pumping are large. Erecinska and Silver,
J.
Cerebr. Blood Flow and Metabolism, 9: 2-19 (1989). In addition, protein
phosphorylation which plays an important role in brain function is also
through to

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consume a sizable fraction of the total energy available in those cells
(Erecinska and
Silver, id. 1989). Finally, CK, together with nerve-specific enolase belongs
to a group
of proteins known as slow component b (SCb). These proteins are synthesized in
neuronal cell body and are directed by axonal transport to the axonal
extremities. Brady
and Lasek, Cell, 23: 515-523 (1981), Oblinger et al., J. Neurol., 7: 433-462
(1987) The
question of whether CK participates in the actual energetics of axonal
transport remains
to be answered.
In conclusion, the CK system plays a key role in the energetics of the adult
brain.
This is supported by 31 P NMR magnetization transfer measurements showing that
the
pseudo first order rate constant of the CK reaction in the direction of ATP
synthesis as
well as CK flux correlate with brain activity which is measured by EEG as well
as by the
amount of deoxyglucose phosphate formed in the brain after administration of
deoxyglucose. The present inventors have discovered that diseases of the
nervous
system can be treated by modulating the activity of the creatine
kinase/creatine
phosphate pathway.
The Role of Creative Kinase in Treating
Diseases of the Nervous System
The mechanisms by which nerve cell metabolites are normally directed to
specific cell tasks is poorly understood. It is thought that nerve cells, like
other cells,
regulate the rate of energy production in response to demand. The creative
kinase
system is active in many cells of the nervous system and is thought to play a
role in the
allocation of high energy phosphate to many diverse neurological processes,
such as
neurotransmitter biosynthesis, electrolyte flux and synaptic communication.
Neurological function requires significant energy and creative kinase appears
to play an
important role in controlling the flow of energy inside specialized excitable
cells such as
neurons. The induction of creative kinase, the BB isozyme and the brain
mitochondria)
creative kinase in particular, results in the generation of a high energy
state which could
sustain or multiply the pathological process in diseases of the nervous
system. Creative
kinase induction also causes release of abnormally elevated cellular energy
reserves
which appear to be associated with certain diseases of the nervous system.
Conversely,
suppression of the creative kinase system, or aberrances in it, induce a low
energy state
which could result in or assist in the death in the process of all the nervous
system.
The components of the creative kinase/phosphocreatine system include the
enzyme creative kinase, the substrates creative and creative phosphate, and
the
transporter of creative. Some of the functions associated with this system
include
efficient regeneration of energy in cells with fluctuating and high energy
demand,

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phosphoryl transfer activity, ion transport regulation, cytoskeletal
association, nucleotide
pool preservation, proton buffering, and involvement in signal transduction
pathways.
The creatine kinase/phosphocreatine system has been shown to be active in
neurons,
astrocytes, oligodendrocytes, and Schwann cells. The activity of the enzyme
has been
shown to be up-regulated during regeneration and down-regulated in
degenerative states,
and aberrant in mitochondria) diseases.
Many diseases of the nervous system are thought to be associated with
abnormalities in an energy state which could result in imbalanced ion
transport
neurotransmitter release and result in cell death. It has been reported that
defects in
mitochondria) respiration enzymes and glycolytic enzymes may cause impairment
of cell
function.
Without wishing to be bound by theory, it is thought that if the induction or
inhibition of creatine kinase is a cause or a consequence of disease,
modulating its
activity, may block the disease. Modulating its activity would modulate energy
flow and
affect cell function. Alternatively, another possibility is that creatine
kinase activity
generates a product which affects neurological function. For example, creatine
phosphate may donate a phosphate to a protein to modify its function (e.g.,
activity,
location). If phosphocreatine is such a phosphate donor, creatine analogs
which are
phosphorylatable or phosphocreatine analogs may competitively inhibit the
interaction
of phosphocreatine with a target protein thereby directly or indirectly
interfering with
nervous system functions. Alternatively, phosphorylatable creatine analogs
with altered
phosphoryl group transfer potential may tie up phosphate stores preventing
efficient
transfer of phosphate to targets. A neurological disease could be associated
with down
regulation of creatine kinase activity. In such cases, replenishment of the
substrates,
e.g., creatine, creatine phosphate or a substrate analog, which could sustain
ATP
production for an extended of time, with other activators of the enzyme could
be
beneficial for treatment of the disease.
Ingestion of creatine analogs has been shown to result in replacement of
tissue
phosphocreatine pools by synthetic phosphagens with different kinetic and
thermodynamic properties. This results in subtle changes of intracellular
energy
metabolism, including the increase of total reserves of high energy phosphate
(see refs.
Roberts, J.J. and J.B. Walker, Arch Biochem. Biophys 220(2): 563-571 (1983)).
The
replacement of phosphocreatine pools with slower acting synthetic phosphagens,
such as
creatine analogs might benefit neurological disorders by providing a longer
lasting
source of energy. One such analog, cyclocreatine (1-
carboxymethyl-2-aminoimidazolidine) modifies the flow of energy of cells in
stress and
may interfere with ATP utilization at sites of cellular work.

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The pathogenesis of nerve cell death in neurodegenerative diseases is unknown.
A significant amount of data has supported the hypothesis that an impairment
of energy
metabolism may underlie the slow exitotoxic neuronal death. Several studies
have
demonstrated mitochondrial or oxidative defects in neurodegenerative diseases.
Impaired energy metabolism results in decreases in high energy phosphate
stores and a
deteriorating membrane potential. Under these conditions the voltage sensitive
Mg2+block of NMDA receptors is relieved, allowing the receptors to be
persistently
activated by endogenous concentrations of glutamate. In this way, energy
related
metabolic defects may lead to neuronal death by a slow exitotoxic mechanism.
Recent
studies indicate that such a mechanism occurs in vivo, and it may play a role
in animal
models of Huntington's disease and Parkinson's disease.
As discussed in detail above, the creative kinase/ creative phosphate energy
system is only one component of an elaborate energy- generating system found
in the
nervous system. The reaction catalyzed by this system results in the rapid
regeneration
of energy in the form of ATP at sites of cellular work. In the mitochondria
the enzyme
is linked to the oxidative phosphorylation pathway that has been implicated in
diseases
of the nervous system. There the enzyme works in the reverse direction where
it stores
energy in the form of creative phosphate.
The invention is further illustrated in the following examples which in no way
should be construed as being further limiting. These examples provide evidence
that
creative compounds, represented by creative itself and the analogue
cyclocreatine, are
neuroprotective agents in animal models used for neurodegenerative diseases,
specifically, Huntington's disease and Parkinson's disease. The contents of
all
references, pending patent applications and published patent applications,
cited
throughout this application {including the background section) are hereby
incorporated
by reference. For example, all teachings with regard to creative compounds,
ATP
enhancing agents, neuroprotective agents, etc. are intended to be part of the
present
invention. It should be understood that the models used throughout the
examples are
accepted models and that the demonstration of efficacy in these models is
predictive of
efficacy in humans.
Examples
Example 1: Models for Huntington's Disease: Malonate and 3-Nitropropionic
Acid
There is substantial evidence that energy production may play a role in the
pathogenesis of neurodegenerative diseases (Beat et al., Ann. Neurol. 31:119-
130
(1992)). Impaired energy production may lead to activation of excitatory amino
acid

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receptors, increases in intracellular calcium and the generation of free
radicals (Beal et
al., Ann. Neurol. 38:357-366 (1995)). In Huntington's Disease (HD) there is
reduced
mitochondrial complex II-III activity in post mortem tissue and increased
cerebral lactate
concentrations in vivo (Browne et al., Ann. Neurol., in press, (1997); Gu et
al., Ann.
Neurol. 39:385-389 (1996); Jenkins et al., Neurology 43 :2689-2695 (1993)).
Animal models of Huntington's disease involve defects in energy production.
Malonate and 3-nitropropionic acid (3-NP) are, respectively, reversible and
irreversible
inhibitors of complex II (succinate dehydrogenase) which produce striatal
lesions similar
to those of HD (Beal et al., J. Neurochem. 61:1147-1150 (1993); Brouillet et
al., PNAS
92:7105-7109 (1995); Henshaw et al., Brain Research 647: 161 -166 (1994)). The
pathogenesis of lesions produced by these compounds involves energy depletion,
followed by activation of excitatory amino acid receptors and free radical
production
(Schulz et al., J. Neurosci. 15:8419-8429 (1995); Schulz et al., J. Neurochem.
64:936-939 (1995)).
The enzyme succinate dehydrogenase plays a central role in both the
tricarboxylic acid cycle and the electron transport chain in the mitochondria.
Intrastriatal
injections of malonate in rats were shown to produce dose dependent striatal
excitotoxic
lesions which are attenuated by both competitive and noncompetitive NMDA
antagonists (Henshaw et al., Brain Res. 647: 161 - 166 (1994)). Furthermore,
the
glutamate release inhibitor lamotrigine also attenuates the lesions. Co-
injection with
succinate blocks the lesions consistent with an effect on succinate
dehydrogenase. The
lesions are accompanied by a significant reduction of ATP levels as well as
significant
increase in lactate levels in vivo as shown by chemical shift resonance
imaging (Beal et
al., J. Neurochem 61:1147-1150 (1993)). Furthermore, the increases in lactate
are
greater in older animals consistent with a marked age of the lesion.
Histological studies
have shown that the lesion spares NADPH-diaphorase neurons. Somatostatin
concentrations were also spared. In vivo magnetic resonance imaging of lesions
shows a
significant correlation between increasing lesion size and lactate production.
A series of experiments demonstrated that the administration of Q 10 or
nicotinimide produced dose dependent protection against the lesions in the
malonate
animal model. These compounds attenuated ATP depletion produced by malonate in
vivo. Furthermore, the co-administration of Q 10 with nicotinimide attenuated
the
lesions and reduced increases in lactate which occurred after intrastriatal
malonate
injections.
All of the above mentioned studies supported malonate and 3-NP as useful
models for
the neuropathologic and neurochemical features of HD. The lesions produced
similar
patterns of cellular sparing seen in HD. There is a depletion of striatal
spiny neurons,

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yet a relative preservation of the NADPH diaphorase interneurons. Furthermore,
there is
an increase in lactate concentration which has been observed in HD.
We initially examined whether oral administration of creative or its analogue
cyclocreatine could attenuate malonate lesions. Creative was administered
orally to rats
in their feed at doses of 0.25-3.0% of the diet. Cyclocreatine was
administered at
0.2-1.0%. Controls received unsupplemented otherwise identical diets. The
compounds
were administered for two weeks prior to the administration of malonate and
then for a
further week prior to sacrifice. Malonate was dissolved in distilled deionized
water and
the pH wad adjusted to 7.4 with 0.1 m HCI. Intrastriatal injections of 1.5 ul
of malonate
containing 3 p,mol were made into the striatum at the level of the Bregma 2.4
mm lateral
to the midline and 4.5 mm ventral to the dura. Animals were sacrificed at 7
days by
decapitation, and the brains were quickly removed and placed in ice cold 0.9%
saline
solution. Brains were sectioned at 2 mm intervals. Slices were then placed
posterior
side down in 2% 2,3,5-triphenyltetrazolium chloride. Slices were stained in
the dark at
room temperature for 30 minutes and then removed and placed in 4%
paraformaldehyde,
pH 7.3. Lesions, noted by pale staining, were evaluated on the posterior
surface of each
section using a Bioquant 4 system by an experienced histologist blinded to
experimental
conditions. These measurements have been validated by comparing them to
measurements obtained on adjacent Nissl stain sections.
In an initial pilot experiment, shown in Figure l, it was found that oral
supplementation with both creative and cyclocreatine protected against
striatal malonate
lesions. A dose response curve for neuroprotection by both creative and
cyclocreatine
against malonate induced striatal lesions was then examined. As shown in
Figure 2,
increasing doses of creative from 0.25-3% in the diet exerted dose dependent
neuroprotective effects against malonate induced striatal lesions. Significant
protection
occurred with doses of 1% and 2% in the diet. There was less protection at 3%
creative,
suggesting that a U shaped dose response may occur with higher doses.
Administration
of cyclocreatine resulted in dose dependent neuroprotective effects which were
significant at a dose of 1 % cyclocreatine.
In the 3-NP model, creative was administered orally at a dose of 1% in feed.
Controls received unsupplemented rat chow. 3-NP was diluted in water and
adjusted to
pH 7.4 with NaOH and administered at a dose of 10 mg/Kg intraperitoneally
every 12
hours. Animals became acutely ill after 9-11 days. Since there was variability
in the
times at which animals became ill, they were clinically examined 3 hours after
the
injections and 1 animal of each group was sacrificed when an animal was
acutely ill,
regardless of whether it was on a control diet or a creative supplemented diet
(Schulz et
al., J. Neurochem. 64:936-939 (1995)). Nine to ten animals were examined in
each

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group. Animals were sacrificed after showing acute illness and striatal lesion
volume
was assessed by TTC staining as above. Statistical comparison was made by
student's t
test.
A remarkable level of neuroprotection was seen against subacute 3-NP
neurotoxicity in creatine treated animals, as shown in Figure 3. Dietary
supplementation
with 1% creatine resulted in significant 83% reduction in lesion volume
produced by
3-NP. This suggests that dietary supplementation with creatine may exert its
greatest
efficacy against more slowly evolving metabolic insults than against acute
insults.
Example 2: MPTP as a model for Parkinson's Disease
MPTP, or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine is a neurotoxin which
produces a Parkinsonian syndrome in both man and experimental animals. The
initial
report was by a chemist who was synthesizing and self injecting an opiate
analogue. He
inadvertently synthesized MPTP and developed profound Parkinsonism. Subsequent
pathologic studies showed severe degeneration in the pars compacta of the
substantia
nigra. A large outbreak subsequently occurred in California. These patients
developed
typical symptoms of Parkinsonism. They also had positron emission tomography
done
which showed a marked loss of dopaminergic innervation of the striatum.
Studies of the mechanism of MPTP neurotoxicity show that it involves the
generation of a major metabolite, MPP+. This metabolite is formed by the
activity of
monoamine oxidase on MPTP. Inhibitors of monoamine oxidase block the
neurotoxicity of MPTP in both mice and primates. The specificity of the
neurotoxic
effects of MPP+ for dopaminergic neurons appears to be due to the uptake of
MPP+ by
the synaptic dopamine transporter. Blockers of this transporter prevent MPP+
neurotoxicity. MPP+ has been shown to be a relatively specific inhibitor of
mitochondria) complex I activity. It binds to complex I at the retenone
binding site. In
vitro studies show that it produces an impairment of oxidative
phosphorylation. In vivo
studies have shown that MPTP can deplete striatal ATP concentrations in mice.
It has
been demonstrated that MPP+ administered intrastriatally in rats produces
significant
depletion of ATP as well as increases in lactate confined to the striatum at
the site of the
injections. The present inventors have recently demonstrated that coenzyme
Q10, which
enhances ATP production, can significantly protect against MPTP toxicity in
mice.
The effect of two representative creatine compounds, creatine and
cyclocreatine,
were evaluated using this model. Creatine and cyclocreatine were administered
in the
initial pilot experiment as 1% formulation in the feed of animals, and was
administered
for three weeks before MPTP treatment. MPTP was administered intraperitoneally
at a
dose of l5mg/kg every 2 hours for five injections. The animals then remained
on either

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creative or cyclocreatine supplemented diets for 1 week before sacrifice. The
mice
examined were male Swiss Webster mice weighing 30-35 grams obtained from
Taconic
Farms. Control groups received either normal saline or MPTP hydrochloride
alone.
MPTP was administered in 0.1 ml of water. The MPTP was obtained from Research
Biochemicals. Eight to twelve animals were examined in each group. Following
sacrifice the two striata were rapidly dissected and placed in chilled 0.1 M
perchloric
acid. Tissue was subsequently sonicated, and aliquots were taken for protein
quantification using a fluorometer assay. Dopamine, 3,4-dihydroxyphenylacetic
acid
(DOPAC), and homovanillic acid (HVA) were quantified by HPLC with 16 electrode
electrochemical detection. Concentrations of dopamine and metabolites were
expressed
as nmol/mg protein. The statistical significance of differences was determined
by
one-way ANOVA followed by Fisher PLSDpost-hoc test to compare group means.
The initial results are shown in Figure 4. Oral administration of either
cyclocreatine or creative significantly protected against DOPAC depletions
induced by
MPTP. Cyclocreatine was effective against MPTP induced depletions of
homovanillic
acid. Both administration of creative and cyclocreatine produce significant
neuroprotection against MPTP induced dopamine depletions. The neuroprotective
effect
produced by cyclocreatine was greater than that seen with creative alone.
A dose response study was conducted where the creative dose was 0.25%3.0% of
the diet and cyclocreatine 0.25-1.0% of the diet. The results, shown in Figure
S,
demonstrate that doses of 0.25%, 0.5% and 1.0% creative exerted dose-dependent
significant neuroprotection effects which disappeared at doses of 2.0% and
3.0%
creative, consistent with a U shaped dose response curve. Cyclocreatine
exerted
significant protection against dopamine depletions at 0.5% and 1.0%
cyclocreatine.
Effects of creative on the dopamine metabolites homovanillic acid (HVA) and
3-4-dihydroxyphenyl acetic acid (DOPAC) paralleled those seen with dopamine.
Cyclocreatine also exerted neuroprotection effects against HVA and DOPAC,
although
protection against HVA depletion was not seen with 0.5% cyclocreatine which we
suspect is due to experimental variability.
These results indicate that the administration of creative or cyclocreatine
can
produce significant neuroprotective effects against MPTP induced dopaminiergic
toxicity. These results imply that these compounds are useful for the
treatment of
Parkinson's disease. The data further establish the importance of the creative
kinase
system in buffering energy and survival of neuronal tissue. Therefore,
creative
compounds which can sustain energy production in neurons are going to emerge
as a
new class of protective agents of benefit therapeutically in the treatment of
neurodegenerative diseases where impairment of energy has been established.

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Example 3: Effect of Dietary Creative in a Mouse Model for ALS
Motor neuron degeneration was generated in transgenic mice that express a
human Cu, Zn superoxide dismutase mutation. Gurney et al., Science, vol. 264,
pp
1772-1775 (1994) These FALS mice develop a syndrome which mimics the symptoms
of familial amyotropic lateral sclerosis (FALS). Gradual loss of motor
function becomes
apparent, and typically the mice do not survive beyond 140 days.
FALS mice were divided into control and test groups. At approximately 80 days
(between 70 and 90 days) after birth, the test groups (containing 5 mice per
group) were
changed over from a standard diet to a diet containing 1 % creative. The
control group
(containing 6 mice per group) were fed the standard diet.
Behavioral Testing-Rotorod
Mice were given two days to become aquatinted with the rotorod apparatus
before testing began. Testing began with the animals trying to stay on a rod
that was
rotating at 1 rpm. The speed was then increased by 1 rpm every 10 seconds
until the
animal fell off. The speed of rod rotation at which the mouse fell off was
used as the
measure of competency on this task. Animals were tested every other day until
they
could no longer perform the task
The results for the test and control animals are shown in Figure 6. As shown
in
the Figure, the creative-fed animals showed significantly better performance
throughout
the experiment suggesting less degeneration of motoneural skills than the
control mice
which were fed a standard diet.
Survival
FALS mice begin to show behavioral symptoms at about 120 days. The initial
symptom is high frequency resting tremor. This progresses to gait
abnormalities and
uncoordinated movements. Later, the mice begin to show hemiparalysis of the
hindlimbs, eventually progressing to paralysis of the forelimbs and finally,
complete
paralysis. Animals in this study were sacrificed when they could no longer
roll over
within 10 seconds of being pushed on their side. This time point was taken as
the time
of death.
The results are shown graphically in Figure 7. Figure 7 shows that the animals
placed on a diet containing 1 % creative survived longer than those placed on
the control
diet. Over 14 days of extension in survival was noted, which is a
statistically significant
improvement over the control mice.
The experiments performed on the FALS mice demonstrate that creative has
beneficial effects as an additional therapy for ALS. It improves the quality
of life and
extends survival.

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Equivalents
Those skilled in the art will recognize, or be able to ascertain using no more
than
routine experimentation, many equivalents to the specific embodiments of the
invention
described herein. Such equivalents are intended to be encompassed by the
following
claims.